The intervention by Vladimir Medinsky follows arson attacks and threats against cinemas.
Mr Medinsky wrote on Twitter that Matilda was just "an ordinary feature film".
The movie, which is yet to be released, has sparked harsh criticism from hard-line nationalists and some Orthodox believers in Russia. They see it as blasphemy against the emperor, who is still greatly revered by the Russian Orthodox Church.
Although most people accept that the affair happened, some Christians maintain its depiction in the movie has been distorted and that the portrayal is vulgar.
Nicholas II was murdered by the Bolsheviks and canonised by the Orthodox Church in 2000.
In August Russian Orthodox Christians protested against the film and gathered to outside a Moscow church, praying for the movie to be banned.
In separate comments to the Tass news agency, Mr Medinsky said "there is nothing (in the film) insulting either for the memory of Nicholas II or for the history of the Russian monarchy".
He called on Russians to "observe the law, common sense and have respect for each other" and urged law enforcement agencies to protect cinemas and audiences.
Russia's largest cinema chain announced on Tuesday it had contacted police about threats it had received over Matilda and would not show the movie because of safety fears.
While Mr Medinsky said the decision by the cinema chain was the prerogative of its owners, he said attempts to pressurise cinemas were "pure lawlessness and censorship", Tass reported.
Two cars were set on fire earlier this week outside the office of the lawyer for director Alexei Uchitel and signs reading "burn for Matilda" were reportedly found near the scene.
Last month, assailants tried to set fire to Mr Uchitel's film studio.
Mr Uchitel has said the audiences who have attended pre-release viewings of Matilda have reacted positively to the film, and has called on the state to ensure the safety of cinema-goers.
Matilda is set to be released in Russia on October 26.
