The US Treasury unveiled a list of 13 current and former officials, including the interior minister, heads of the national police, army and national guard, the president of the electoral council, and the finance chief of state oil company PDVSA, whose US assets would be frozen.
The announcement came as Venezuela's opposition launched its nationwide stoppage as part of a campaign to halt Sunday's vote and, more broadly, force Maduro from office through early elections.
But in a country suffering from widespread shortages of basic goods and soaring inflation, protesters are showing their discontent with Maduro's leadership.
"No more dictatorship!" read signs on road barricades in eastern Caracas.
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But the left-wing leader has been defiant, saying the foreshadowed threat by US President Donald Trump to impose sanctions made him more determined than ever to forge on with his plan.
Maduro accuses the US of fomenting the unrest against him and his government, with the help of the conservative opposition.
But some 70 per cent of Venezuelans are opposed to the Constituent Assembly, according to polling firm Datanalisis.
The hardening political struggle has deepened fears that street violence that has erupted in Venezuela during anti- Maduro protests over the past four months could worsen. Already more than 100 people have died.
The opposition has planned another major demonstration in the capital on Friday.
Thousands of Venezuelans loaded with heavy bags have crossed the border into Colombia this week, fleeing the unrest.
"The elections are on Sunday and we really don't know what will happen," said one, Maria de los Angeles Pichardo, who left with her husband and son. "To be safe, we prefer to cross."
"Every time we're worse off, with long lines and shortages. I think I'll strike for 48 hours," said one Caracas resident, Maria Auxiliadora.
Prominent opposition leader Leopoldo Lopez made a direct appeal to the military early today to withdraw its support from Maduro's plan, which he called a "constitutional fraud" aimed at eliminating democratic rule.
In announcing the US sanctions, Treasury Secretary Steven Mnuchin said America was "standing by the Venezuelan people in their quest to restore their country to a full and prosperous democracy."
The opposition, bolstered by an unofficial vote on July 16 that saw a third of the electorate reject Maduro's plan, has called for a boycott of Sunday's vote.
The president's attorney general, Luisa Ortega, has also broken ranks with the government over the issue and become a vociferous opponent.
At the same time, Maduro's administration is being squeezed by a long-running economic crisis.
The oil export-dependent economy will shrink 12 per cent this year, after a contraction of 18 per cent last year, according to the latest forecast from the International Monetary Fund.
Venezuela's currency reserves have dwindled to under USD 10 billion as the government keeps up debt repayments at the expense of imports to stave off a crippling default.
The opposition-led National Assembly, meanwhile, has challenged the government by appointing 33 supreme court judges to rival ones loyal to Maduro.
Three of the "shadow" judges have been arrested in the past few days by Venezuelan intelligence officials.