National Elections Council president Francisco Carrasquero cited a series of procedural errors in denying the petition which was backed by more than three million signatures.
The rejection was a blow to opposition hopes to try to vote the populist president out of office this year.
But opposition leaders have already said they will make a second nationwide collection of signatures to seek a referendum.
The decision was likely to re-ignite political tensions in the oil-rich South American nation, which has been shaken for more than a year by fierce political feuding over the rule of Chavez.
Before the electoral body's decision, several thousand Chavez supporters carrying banners reading "No Referendum with Dud Signatures," staged a protest in Caracas.
Chavez, who himself led a botched coup six years before winning a landslide election in 1998, is resisting the referendum challenge.
Leading a noisy campaign by his supporters against the vote, he had argued the opposition signatures were riddled with forgeries.
Chavez (R) is a fan of Fidel Castro
They were collected on 2 February at the end of a two-month opposition general strike that the president said aimed to topple him unlawfully.
Pilloried by his foes as an authoritarian ruler trying to mimic Cuba's communist president, Fidel Castro, Chavez survived a brief military coup last year.
He dismisses his foes as rich "oligarchs" who oppose his self-styled "revolution" because it threatens their wealth and privileges.