The characters of Paradise Regained center around the account of Satan tempting Jesus Christ, according to the Gospel of Luke. There are also references to other Biblical books in the poem. The two main characters of Milton's epic poem are Jesus Christ and Satan.

Milton also incorporates additional characters, some briefly and some throughout the text, including God, the Father; Mary, the mother of Jesus; Joseph, the husband of Mary; as well as Adam and Eve. Other Biblical characters are Gabriel, the angel; John the Baptist, the prophet who baptized Jesus Christ; David, shepherd and king in the Old Testament; prophets Simeon and Anna; and King Ahab. Milton also references general figures in the Bible, such as angels and demons, Jewish temple leaders, and the wise men who came to visit Christ after his birth.

Additional Biblical characters Milton mentions are Andrew and Simon, followers of Christ; Moses, leader of the Jewish people; Solomon, the wise king; the prophets Elijah and Daniel; Gideon, a leader of Jews; Jephtha, a judge; Abraham, a leader of Jews; and Jacob, a leader of Jews.

In addition to Biblical characters, Milton incorporates characters from Paradise Lost, such as the demon Belial and the dark angel Asmodai. Milton also references the demon Incubus, as well as various goddesses and gods, like Neptune.

This epic poem also mentions historical figures, such as Julius Caesar, Socrates, Tiberius, Alexander the Great, and Homer.