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Isaac (Yitschaq, Yischaq) is mentioned by name 80 times in the King James Version of Genesis, 32 times in the remainder of the Hebrew Bible, and 20 times in the New Testament. In the narrative, God calls Isaac the "only son" of Abraham (Gen. 22:12, 22:16, cf. Heb. 11:17), though Abraham's sons also include Ishmael and six others. Variations of the formula "Abraham, Isaac, and Jacob" occur 23 times in the Hebrew Bibl Isaac is first prophesied at Gen. 12:7, is born at 21:2, dies at 35:29, and is remembered at 50:24. According to the documentary hypothesis, use of names of God indicates authorship, and form critics variously assign passages like 26:6–11 to the Yahwist source, and 20:1–7, 21:1–22:14, and 22:19 to the Elohist source; this source-critical approach has admitted problems, in that the name "Yahweh" appears in Elohist material. According to the compilation hypothesis, the formulaic use of the word toledoth (generations) indicates that Gen. 11:27–25:19 is Isaac's record through Abraham's death (with Ishmael's record appended), and Gen. 25:19–37:2 is Jacob's record through Isaac's death (with Esau's records appended).

When Sarah was beyond child-bearing age, God told Abraham and Sarah that she would still give birth, at which she privately laughed (Gen. 18:10–12). Isaac was born when Abraham was 100, and Abraham circumcised Isaac when the boy was eight days old (21:1–5). Isaac was Sarah's first and only child, but Abraham had had another son, Ishmael, thirteen years earlier, borne by Sarah's maidservant, Hagar (16:15). After Isaac had been weaned, Sarah saw Ishmael mocking, and urged her husband to banish Hagar and Ishmael so that Isaac would be Abraham's only heir. Abraham was hesitant, but at God's order he listened to his wife's request (21:8–12).

Later, God tested Abraham by commanding him to sacrifice his son.Abraham obeyed and took Isaac to mount Moriah. Without murmuring, Isaac let Abraham bind him and lay him upon the altar as a sacrifice. Abraham took the knife and raised his hand to kill his son; at the last minute, the angel of the LORD prevented him from doing so. Instead of Isaac, Abraham sacrificed a ram that was trapped in a thicket nearby. (According to Islam, the Biblical narrative is incorrect and these events happened to Ishmael instead.)

When Isaac was 40, Abraham sent Eliezer, his steward, into Mesopotamia to find a wife for Isaac, from his nephew Bethuel's family. Eliezer chose Rebekah for Isaac. After many years of marriage to Isaac, Rebekah had still not given birth to a child and was believed to be barren. Isaac prayed for her and she conceived (25:20–21). Rebekah gave birth to twin boys, Esau and Jacob. Isaac was 60 years old when his two sons were born (25:24–26). Isaac favored Esau, and Rebekah favored Jacob.

As Abraham grew rich, Isaac stayed with his father, and was about 75 when his father died. Like Abraham, Isaac deceived Abimelech about his wife, and Isaac built his business by digging wells, unstopping those that his father had dug and the Philistines had stopped up. Isaac had a vision of God at Beersheba and made a treaty with Abimelech there.

Isaac grew old and became blind. He called his son Esau and directed him to procure some venison for him, in order to receive Isaac's blessing. While Esau was hunting, Jacob deceptively misrepresented himself as Esau to his blind father and obtained his father's blessing, making Jacob Isaac's primary heir, and leaving Esau in an inferior position. Isaac sent Jacob into Mesopotamia to take a wife of his own family. After 20 years working for Laban, Jacob returned home, and he and Esau buried Isaac when Isaac died at the age of 180 (35:28–29).