Independent politician Yekaterina Duntsova wanted to run on a platform to end the war with Ukraine.
But the electoral commission voted unanimously to reject her candidacy three days after her application, citing 100 "mistakes" on her form.
Ms Duntsova said she would appeal the decision at the Supreme Court.
The presidential election, which will be held in March 2024, is Russia's first since President Putin launched his full-scale invasion of Ukraine nearly two years ago.
The immediate slap-down of a Putin critic will be seen as evidence by some that no dissent will be tolerated in the campaign.
The head of Russia's electoral commission, Ella Pamfilova, said Ms Duntsova would not be allowed to progress to the next stage of gathering thousands of supporters' signatures.
"You are a young woman, you have everything ahead of you. Any minus can always be turned into a plus. Any experience is still an experience," she told Ms Duntsova, 40, after the decision.
The former TV journalist had declared she would run for the presidency in November. At the time, she told the Reuters news agency: "Any sane person taking this step would be afraid - but fear must not win."
Russia's constitution was amended in 2020, increasing the presidential term from four to six years and giving Mr Putin a clean slate to run again by cancelling out his previous terms.
Moscow has sidelined opposition figures for years, and President Putin is expected to win in March; the Kremlin claims he enjoys genuine support among Russians.
Ms Duntsova had been vocal on her plans to run a campaign on ending the war in Ukraine and freeing political prisoners.
She was quick to respond after the commission's decision. "We will appeal to the Supreme Court, since this decision is not based on the law," a message on her Telegram channel said.
