New York-born Martelly had provided election authorities with documents showing she had renounced her American citizenship.
But the National Bureau of Electoral Disputes, or BCEN, said Martelly's documentation was insufficient to confirm her sole Haitian nationality.
"We are shocked," Martelly's lawyer Gregory Mayard-Paul told AFP yesterday.
"This is a shameful vote that is not based in electoral law. On top of everything else, it is a violation of Sophia Saint-Remy Martelly's civil rights."
President Michel Martelly's wife announced her Senate candidacy on April 23.
The Haitian Constitution, amended in 2012, recognises dual nationality.
But an electoral decree published in March of this year still requires candidates for any election in the country to have never renounced Haitian nationality, or to have held any other nationality when registering for the vote.
The opposition rejoiced at the news that the first lady was barred from the election.
Political analyst Chantal Elie welcomed the move as a sign that election authorities were independent.
"The fight for the rule of law continues today, tomorrow and always... Let's not lose hope! Victory is near," she wrote on Twitter.
More than 6,000 posts are up for grabs in elections due to be held August 9, October 25 and December 27, including for president, parliament and local municipal governments.
It is a huge logistical challenge for the desperately poor nation.
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