"I assert that Putin is behind this act, I don't see any other explanation," he told German news magazine Der Spiegel in an interview.
Mr Navalny is recovering in Germany after becoming ill on a flight in Siberia in August. Germany says he was poisoned by a Novichok nerve agent, findings which were confirmed by labs in France and Sweden.
The Kremlin denies any involvement.
Mr Putin's spokesman said there was no evidence that Mr Navalny had been poisoned with a nerve agent, and said CIA agents were working with the opposition leader.
Mr Navalny collapsed on a flight in Russia's from Tomsk to Moscow on August 20. The plane made an emergency landing in Omsk, where he spent two days in a coma before being transferred by a medical jet to the Charité hospital in Berlin.
In his first interview since he fell ill, published in Der Spiegel on Thursday, Mr Navalny, 44, said the order to use Novichok could only have come from the heads of three of Russia's intelligence services, who all work for Vladimir Putin.
"If 30 people have access to a [chemical] agent, and not three, then it's a global threat," he told the magazine.
Mr Navalny's supporters initially believed his tea had been spiked at Tomsk airport. However, traces of Novichok were later found on water bottles at the hotel where he had stayed the night before.
Speaking of his ordeal, Mr Navalny said: "You feel no pain, but you know you're dying. Straight away."
He said he had only been able to receive medical care because of "a chain of lucky circumstances".
“I believe they were determined not to let me leave the country, and that is why they publicly declared I was not in a fit state to be transported,” Navalny said. “They were waiting for me to die. But because of support for me and the efforts of my wife, the whole thing turned into a kind of reality show, with the title ‘Navalny dies in Omsk’ .
“For Putin’s people it is important that they don’t lend their enemies a victim status. If I had died in Omsk or left with permanent damage there, then the responsibility would have clearly been with them.”
Asked why the Russian president would target him, Mr Navalny suggested that Mr Putin may have targeted him to avoid a "Belarus situation", referencing the weeks of mass anti-government protests in Belarus which followed a disputed election.
"The system is fighting for its survival and we've just felt the consequences."
Mr Navalny was released from the Charité hospital in Berlin last week. He is undergoing physiotherapy to help his recovery.
His spokeswoman said last week that his bank accounts had been frozen and his flat in Russia seized.
However, Mr Navalny told Der Spiegel he still intended to return to Russia.
"Not going back would mean that Putin had achieved his goal," he said. "I will not give Putin the gift of not returning to Russia."
The EU and several governments have called for Russia to investigate Mr Navalny's poisoning.
Russian ex-spy Sergei Skripal and his daughter Yulia were poisoned by a nerve agent from the Novichok group in Salisbury in 2018. Although they both survived, a local woman, Dawn Sturgess, died after coming into contact with the poison.
Moscow denied any involvement in the poisoning. However, Britain accused Russia's military intelligence of carrying out that attack, and twenty countries expelled more than 100 Russian diplomats and spies.