Theresa May said the diplomats, who have a week to leave, were identified as "undeclared intelligence officers".
The UK later told the UN Security Council that Russia had used "a weapon so horrific that it is banned in war" in a "peaceful" British city.
Russia denies attempted murder and says it will respond appropriately.
Mrs May also revoked an invitation to Russia's foreign minister, and said the Royal Family would not attend the Fifa World Cup in Russia later this year.