Poythress lived on his family farm in Madera, California until he was five years old and later moved with his family to Fresno, California. In 1981, he was ordained as a teaching elder in the Reformed Presbyterian Church, Evangelical Synod, which merged into the Presbyterian Church in America. He married his wife Diane in 1983, and they have two children, Ransom and Justin.

He earned a B.S. in Mathematics from California Institute of Technology (1966), where he was a Putnam fellow in 1964, and a Ph.D. in mathematics from Harvard University (1970). He studied linguistics and Bible translation at the Summer Institute of Linguistics at the University of Oklahoma in 1971 and 1972, and he enrolled at Westminster Theological Seminary, earning an M.Div. (1974) and a Th.M. in apologetics (1974). He then received an M.Litt. in New Testament from University of Cambridge (1977) and a Th.D. in New Testament from the University of Stellenbosch, Stellenbosch, South Africa (1981).

He taught mathematics at Fresno State College (now California State University, Fresno) for a year after earning his Ph.D. in the subject, and he taught linguistics at the Summer Institute of Linguistics in the summers of 1974, 1975, and 1977. He has taught New Testament at Westminster Theological Seminary in Philadelphia since 1976.

As an evangelical Poythress advocates the complete, divine authority of Bible, and as a Calvinist he places an emphasis on the sovereignty of God and adopts the Neo-Calvinist notion that Jesus is Lord of every area of life, not just private, religious life. He makes use of Biblical Theology in the tradition of Geerhardus Vos, and builds on Meredith G. Kline's work in Images of the Spirit to argue that "imaging" is a pattern in the Bible beyond man and woman being made in the image of God. He has a decidedly positive view of the Old Testament Law, though he rejects theonomy, and he rejects the hermeneutics of dispensationalism. In Christian eschatology, he advocates an Amillennial view (compare the summary of Christian eschatological differences).

He is an advocate of Cornelius Van Til's presuppositional apologetics, particularly the ideas that epistemology and ontology must find their ultimate grounding in the Trinity. He has also sought to work out presuppositionalism's central claim that there is no neutrality in the area of science and mathematics. In a manner akin to Augustine's view that truth is divine, Poythress views scientific law as a form of the word of God. In 1976, Poythress broke new ground with an chapter on "A Biblical View of Mathematics," while in a 1983 article, he argued that mathematics is the rhyme of the universe.

A central idea in Poythress' thought has concerned the validity of multiple perspectives, or multiperspectivalism, a project that he shares with his teacher and collaborator John Frame. In Poythress's seminal work Philosophy, Science, and the Sovereignty of God, he explored how the scientific concepts of wave, particle and field can be used analogically to demonstrate different ways of looking at things. He argued that such a triadic structure is a "a means of avoiding unhealthy dualism", and he continued on this line of thought in Symphonic Theology, where he applied multiperspectivalism to theology.

Poythress has published a number of books in different fields — Christian philosophy of science, linguistics, theological method, dispensationalism, biblical law, copyright law, hermeneutics, Bible translation, and eschatology and the Book of Revelation, many of which he has made available online for free:

• Philosophy, Science, and the Sovereignty of God. Presbyterian and Reformed: 1976, reprinted 2004. ISBN 978-1596380028.
• Symphonic Theology: The Validity of Multiple Perspectives in Theology. Zondervan: 1987. ISBN 978-0875525174.
• Understanding Dispensationalists. 2d ed. Presbyterian and Reformed: 1993. ISBN 978-0875523743.
• Science and Hermeneutics: Implications of Scientific Method for Biblical Interpretation (Foundations of Contemporary Interpretation series, volume 6). Zondervan: 1988. ISBN 978-0310409717.
• The Shadow of Christ in the Law of Moses, Wolgemuth & Hyatt: 1991, reprinted Presbyterian & Reformed: 1995. ISBN 978-0875523750.
• God-Centered Biblical Interpretation. Presbyterian and Reformed: 1999. ISBN 978-0875523767.
• The Returning King: A Guide to the Book of Revelation, Presbyterian and Reformed: 2000. ISBN 978-0875524627.
• The Gender-Neutral Bible Controversy: Muting the Masculinity of Gods Words (with Wayne Grudem). Broadman and Holman: 2000. ISBN 978-0805424416.
• Redeeming Science: A God-Centered Approach. Crossway Books: 2006. ISBN 978-1581347319.
• In The Beginning Was The Word: Language--A God-Centered Approach. Crossway Books: 2009. ISBN 978-1433501791.

He has been editor of the Westminster Theological Journal since 2005, and he has contributed to a number of other volumes such as The Foundations of Christian Scholarship, Hermeneutics, Inerrancy, and the Bible, The New Testament Student and His Field, Inerrancy and Hermeneutic: A Tradition, A Challenge, A Debate, Theonomy: A Reformed Critique, Recovering Biblical Manhood and Womanhood, New Geneva Study Bible, and The Spirit of the Reformation Study Bible. He was also a member of the Translation Oversight Committee for the English Standard Version.

Additionally, he has published a number of scholarly articles, including:

• "A Biblical View of Mathematics," in Gary North (ed.), Foundations of Christian Scholarship (Vallecito, CA: Ross House, 1976), 159-188.
• "Structuralism and Biblical Studies," Journal of the Evangelical Theological Society 21, 1978.
• "Science as Allegory," Journal of the American Scientific Affiliation 35, 1983.
• "Newton's Laws as Allegory," Journal of the American Scientific Affiliation 35, 1983.
• "Mathematics as Rhyme," Journal of the American Scientific Affiliation 35, 1983.
• "The Use of the Intersentence Conjunctions De, Oun, Kai, and Asyndeton in the Gospel of John," Novum Testamentum 26, 1984.
• "Reforming Ontology and Logic in the Light of the Trinity: An Application of Van Til's Idea of Analogy," Westminster Theological Journal 57, 1995.
• "2 Thessalonians 1 Supports Amillennialism," Journal of the Evangelical Theological Society 37, 1995.
• "Counterfeiting in the Book of Revelation as a Perspective on Non-Christian Culture," Journal of the Evangelical Theological Society 40, 1997.
• "Keep on Praying!" Decision, 39/10, 1998.
• "Why Scientists Must Believe in God: Divine Attributes of Scientific Law," Journal of the Evangelical Theological Society 46, 2003.