Investigators also opened a criminal investigation into obstructing the work of election officials after Navalny's allies and ordinary Muscovites staged a series of pickets and rallies in the Russian capital in recent days.
For that offence the protest leaders could face up to five years in prison.
A coordinator of Navalny's Moscow headquarters, Oleg Stepanov, said he had also been detained.
The latest crackdown on Russia's opposition politicians came as they have fought to get on the ballot for the Moscow parliament elections in September amid falling approval ratings for President Vladimir Putin.
They have however been disqualified on what critics say are spurious reasons.
Navalny and other anti-Kremlin politicians threatened to stage a major rally on July 27, near the mayor's office, unless Moscow's authorities registered opposition candidates within seven days.
- 'Detained in shorts' -
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On Wednesday, Navalny said he was detained as he was leaving his Moscow home to go jogging and buy flowers for his wife's birthday.
"People are right when they say that sport is not always good for your health," Putin's top opponent quipped.
"I have been detained and am now at a police station wearing shorts like a stupid man," the 43-year-old said.
His spokeswoman Kira Yarmysh said Navalny faced up to 30 days in prison for calls to stage an "unauthorised" rally.
Navalny has already served a 10-day jail sentence for allegedly violating a protest law earlier this month.
Navalny's arrest came after more than 22,000 people rallied in the Russian capital last Saturday to demand independent politicians be allowed to run in the September 8 vote.
The opposition said Saturday's authorised protest was the largest since 2012, when tens of thousands rallied against election fraud during parliamentary polls.
The would-be opposition candidates have also been meeting with supporters and staging pickets outside Moscow City Hall over the past week.
Investigators said in a statement they would "question the organisers and participants of unauthorised rallies and pickets".
The protests involved "threats to use violence against members of the electoral commissions", the Investigative Committee said in a statement.
The opposition politicians have worked hard to get on the ballot paper as they seek to capitalise on anger over declining living standards and unchecked corruption.
They say they were made to jump through countless hoops, and each had to collect roughly 5,000 signatures to be eligible.
But electoral authorities still refused to register the representatives of the opposition, accusing them of faking some of the signatures and refusing to review evidence that they were authentic.
- 'City Hall is afraid' -
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The Kremlin dismissed the opposition's complaints but many Russians are furious at what they perceive as electoral interference.
After the protest, 17 independent candidates including Ilya Yashin and Lyubov Sobol issued a joint statement, accusing Moscow mayor Sergei Sobyanin of sparking a "political crisis" in the city of some 15 million people.
On Tuesday, the disqualified politicians met with the country's election chief, Ella Pamfilova, who admitted that the situation was "unfair".
The talks however led nowhere, the opposition said, accusing Pamfilova of trying to play for time.
Yashin expressed hope that Navalny's arrest would "mobilise" people.
Another independent would-be candidate, Konstantin Jankauskas, said the crackdown was an attempt to discourage people from attending the Saturday rally.
"It means City Hall is afraid that a lot of people would turn up," he said on Twitter.
Police this week warned Muscovites against taking part in an "unauthorised rally", saying their safety might come under threat.
Political commentator Alexander Kynev said the authorities would seek to nip in the bud the new protest wave by launching a fresh crackdown.
"If this does not help and a lot of people turn up on Saturday then they'll think what to do next," he told AFP.