The expression "separation of church and state" in American politics is from Jefferson's response to the Danbury Baptist Association (of Connecticut), in which he reassured them that the First Amendment meant that other larger religious groups would not be permitted to use the power of the federal government to oppress Baptists.
Religious persecution had been a live issue in New England, where the Congregationalist Puritans of the Massachusetts Bay Colony had earlier expelled Baptists, Quakers, and other religious nonconformists from the colony.
You can read the full text of the Danbury Baptists' letter to Jefferson, and Jefferson's response, here on Wikipedia.
To summarize greatly, the Baptists say "We believe in religious liberty, but we've seen persecution before; and we worry that the federal government will be used to impose someone else's religious views on us. We want a government that only punishes people for harming others, and can't be pressured into imposing religious laws."
And Jefferson says "Yes, that's why we put this First Amendment thing in; to build a wall of separation between church and state."
This goes along with someone else's question above in response to Boebert - "which church?" It's ironic that the biggest benefactors of separation are religions, and they're the first to try and dismantle it.
The founders of the Constitution and others back then disagreed on a lot of issues and had a few false starts, as well as expected us to continue to modify laws as society changed. But they certainly agreed on no religion within politics, they lived in a world where that had been shown to be problematic over and over. We can see it now as religion creeps back in to our current politics as well as worldwide in other nations.