Born in Leghorn, Montefiore was brought up in London, where he was taught elementary Hebrew by his mother's brother. First apprenticed to a firms of grocers and tea merchants, he left to become one of the 12 "Jew brokers" in the City of London. After initial setbacks, he went into partnership with his brother Abraham and they established a fine reputation.

In 1812, Montefiore married Judith Cohen, which made him Nathan Mayer Rothschild's brother-in-law and stockbroker. He retired from business in 1824 and devoted his time and resources to community and civic affairs.

His first visit to Eretz Yisrael had a profound religious effect on him, and from then until the end of his life, he became strictly observant. In all, he visited Eretz Yisrael seven times. He established his own synagogue on his estate at Ramsgate and in later years, traveled with his own "shohet" (charged with slaughtering animals in accordance with Jewish law).

His early activities on behalf of the Jews in Eretz Yisrael included a plan to acquire land to help Jews become self-sufficient, as well as attempting to bring industry to the country by introducing a printing press and a textile factory. He inspired the founding of several agricultural settlements as well as Yemin Moshe outside of Jerusalem's Old City which was named after him.

Montefiore was Sheriff of London, 1837-1838, and was knighted by Queen Victoria. He received a baronetcy in 1846 in recognition of his humanitarian efforts on behalf of the Jews. He was president of the British Board of Deputies from 1835-1874, with one brief interruption. Despite his position, he did not play a prominent role in the emancipation struggle, preferring to helped oppressed Jewish communities abroad.

He was known to have such stature that he visited Russia in 1846 to ask the authorities to stop persecution of the Jews. He also visited Morocco in 1863 and Romania in 1867 for the same purpose. Montefiore deeply loved Eretz Yisrael and believed in its messianic restoration as opposed to the large-scale, planned development of the country as the solution to the Jewish problem.

Sir Moses Montefiore's physical stature (he was 6 ft. 3 in. tall), together with his background and his philanthropy, made him highly respected and admired in England and abroad. His 100th birthday was a public holiday in Jewish communities around the world.