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The Codex Vaticanus (The Vatican, Bibl. Vat., Vat. gr. 1209; no. B or 03 Gregory-Aland, δ 1 von Soden), is one of the oldest extant manuscripts of the Greek Bible (New Testament), one of the four great uncial codices. The Codex is named after its place of conservation in the Vatican Library, where it has been kept since at least the 15th century. It is written on 759 leaves of vellum in uncial letters and has been dated palaeographically to the 4th century. The manuscript became known to Western scholars as a result of correspondence between Erasmus and the prefects of the Vatican Library. Portions of the codex were collated by several scholars, but numerous errors were made during this process. The Codex's relationship to the Latin Vulgate was unclear and scholars were initially unaware of the Codex's value. This changed in the 19th century when transcriptions of the full codex were completed. It was at that point that scholars realised the text differed significantly from the Vulgate and the Textus Receptus. Current scholarship considers the Codex Vaticanus to be one of the best Greek texts of the New Testament, with the Codex Sinaiticus as its only competitor. Until the discovery by Tischendorf of the Sinaiticus text, the Codex was unrivaled. It was extensively used by Westcott and Hort in their edition of The New Testament in the Original Greek in 1881. The most widely sold editions of the Greek New Testament are largely based on the text of the Codex Vaticanus.

Codex Vaticanus originally contained a virtually complete copy of the Septuagint ("LXX"), lacking only 1-4 Maccabees and the Prayer of Manasseh. The original 20 leaves containing Genesis 1:1–46:28a (31 leaves) and Psalm 105:27–137:6b have been lost and were replaced by pages transcribed by a later hand in the 15th century. 2 Kings 2:5–7, 10-13 are also lost because of a tear to one of the pages. The order of the Old Testament books in the Codex is as follows: Genesis to 2 Chronicles as normal; 1 Esdras; 2 Esdras (Ezra-Nehemiah); the Psalms; Proverbs; Ecclesiastes; Song of Songs; Job; Wisdom; Ecclesiasticus; Esther; Judith; Tobit; the minor prophets from Hosea to Malachi; Isaiah; Jeremiah; Baruch; Lamentations and the Epistle of Jeremiah; Ezekiel and Daniel. This order differs from that followed in Codex Alexandrinus. The extant New Testament of the Vaticanus contains the Gospels, Acts, the General Epistles, the Pauline Epistles, and the Epistle to the Hebrews (up to Hebrews 9:14, καθα[ριει); it is lacking 1 and 2 Timothy, Titus, Philemon, and Revelation. These missing leaves were supplemented by a 15th-century minuscule hand (folios 760–768) and are catalogued separately as the minuscule Codex 1957. Possibly some apocryphal books from the New Testament were included at the end (as in codices Sinaiticus and Alexandrinus), as it is also possible that Revelation was not included.

The manuscript is in quarto volume, arranged in quires of five sheets or ten leaves each, similar to the Codex Marchalianus or Codex Rossanensis; but unlike the Codex Sinaiticus which has an arrangement of four or three sheets. The number of the quires is often found in the margin. Originally it must have been composed of 830 parchment leaves, but it appears that 71 leaves have been lost. Currently, the Old Testament consists of 617 sheets and the New Testament of 142 sheets. The parchment is fine and thin. The actual size of the pages is 27 cm by 27 cm; although the original was bigger. The codex is written in three columns per page, with 40–44 lines per page, and 16–18 letters per line. In the poetical books of the Old Testament (OT) there are only two columns to a page. In Pentateuch, Joshua, Judges, Ruth, and 1 Kings 1:1–19:11 there are 44 lines in a column; in 2 Chronicles 10:16–26:13 there are 40 lines in a column; and in the New Testament always 42.