Born in Tel Aviv, Gafni was educated in a yeshiva, and later moved to and worked as head of a Kollel in Ofakim. He lives in Bnei Brak, is married, and has three children. ^{[citation needed]}

He was first elected to the Knesset on Degel HaTorah's list in 1988, and was appointed Deputy Minister of Religious Affairs in Yitzhak Shamir's government in 1990. For the 1992 elections, the party joined Agudat Yisrael in forming an alliance called United Torah Judaism, which won four seats. Although he initially lost his seat, Gafni entered the Knesset in 1994 as part of a rotation agreement between the two factions. A similar arrangement operated after the 1996 elections, with Gafni taking the seat for the first half of the session (i. e., until 1998).

Early elections in 1999 meant that Gafni re-appeared in the Knesset sooner than expected. This time, no rotation agreement was in place, so he served his full term, and was re-elected in both 2003 and 2006. When Degel HaTorah split from Agudat Yisrael during the latter stages of the 16th Knesset, Gafni was appointed the party's Parliamentary Group Chairman.

In the 18th Knesset, Gafni was the chairman of the Knesset's financial committee.

Gafni was strongly opposed to the Supreme Court ruling that the state must recognize gay marriages carried out abroad, stating: "We don't have a Jewish state here. We have Sodom and Gomorrah here."^[1] However, he was one of the few ultra-Orthodox public figures to condemn the violence carried out by members of the community over plans for the 2006 Jerusalem gay pride parade.^[2]

In February 2016, Israeli Prime Minister Benjamin Netanyahu criticized Gafni and other government leaders for making disparaging remarks about Reform and Conservative Judaism. Gafni, following a decision to expand the egalitarian section of the Western Wall, declared he would refuse to recognize the decision, and that Reform Jews were "a group of clowns who stab the Holy Torah".^[3]

In 2023 Gafni and Ya'akov Asher submitted a bill that would have banned Proselytizing of Christianity in Israel.^[4][5][6][7]

• Moshe Gafni on the Knesset website