Senate Speaker Renan Calheiros rejected the attempt by Waldir Maranhao, the lower house's acting speaker, to halt the process.
Mr Maranhao had called for a new vote in the lower house.
But to boos and cheers in the Senate, Speaker Renan Calheiros called that decision illegal.
The Senate is scheduled to vote on Wednesday on whether to start an impeachment trial.
The president of the Senate impeachment commission also said the vote would take place as scheduled.
If Ms Rousseff loses, she will be suspended from office, pending a trial that could last six months. She faces allegations that her government violated fiscal rules.
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In his decision, Mr Maranhao said there had been irregularities during the lower house session in which its members overwhelmingly voted in favour of the impeachment process going ahead.
He said members of the lower house should not have publicly announced what their position was prior to the vote, and that it had been wrong of party leaders to instruct their members how to vote.
Mr Maranhao called for a new vote in the lower house.
But Mr Calheiros said in a special session that he would ignore Mr Maranhao's order, and go ahead with the Senate vote. He accused Mr Maranhao of "toying with democracy".
'Political fiasco', Wyre Davies, BBC News South America correspondent
Demonstrator in Brazil shows sign in favour of impeachmentIWhen I sat down for an extended interview with Dilma Rousseff in Brasilia last week, she was fully prepared for and anticipating her likely suspension as president. She assumed, like just about everyone else in Brazil, that the country's Senate would vote in favour of a full impeachment trial.
Indeed, Ms Rousseff told me she would fight to clear her name after her expected suspension from office later this week over charges that she illegally hid the scale of the budget deficit. The decision by the interim speaker of the lower house to annul the impeachment process took everyone - including the president - by surprise.
Whatever the outcome - and don't bet against the impeachment process getting "back on track" - this fiasco does the image of Brazil and its discredited political system no good.
An increasing majority of Brazilians are not just dissatisfied with the country's situation in general but, in particular, with the behaviour of politicians in Brasilia. The place is, rightly or wrongly, perceived to be riddled with corruption where politicians serve only in their self-interest while the rest of the country struggles with a worsening economy, an ever more violent society and with the country's image being ridiculed overseas.
Mr Maranhao, who opposed the impeachment process in the 17 April vote, only took over as the speaker of the lower house last week, after the previous speaker, Eduardo Cunha, was suspended.
Mr Cunha, an outspoken critic of President Rousseff, led the impeachment drive against her.
Ms Rousseff has said the efforts to impeach her amount to "a coup attempt".
She has accused Mr Cunha and Vice-President Michel Temer of being the "ringleaders of the coup".
Brazilian Vice President Michel TemerImage copyrightAPImage caption
Mr Temer would take over as Brazil's interim president if Ms Rousseff were suspended
Mr Temer would step in as interim president if Ms Rousseff were to be suspended from office.
In a BBC interview last week, Ms Rousseff said she was an "innocent victim" and that she would fight on.
She is accused of manipulating the government budget ahead of her re-election in 2014. The president has defended her fiscal measures as common practice in Brazil.
Investors have criticised Ms Rousseff and her Workers' Party for what they say are interventionist policies and see Mr Temer as being more market-friendly.
Brazil is in its worst recession in 25 years, with inflation at a 12-year-high in 2015.
The president's approval ratings have plummeted recently, and recent polls suggest most Brazilians support her removal from office.
Dilma Rousseff attended a university ceremony in Brasilia on Monday while the confusion was ongoing