Lamido Sanusi told a senate committee on Tuesday that out of $67bn of oil sold between January 2012 and July 2013, $20bn was still missing.
The row over the Nigerian National Petroleum Corporation's accounts began late last year following a leaked memo.
Correspondents say the large sums at stake have shocked Nigerians.
Nigeria is Africa's largest oil producer, but the vast majority of its 166 million inhabitants live on less than $2 a day.
'Little understanding'
In Mr Sanusi's initial memo to President Goodluck Jonathan in September, he alleged that about $50bn was missing.
The government admitted that $10.8bn had yet to be remitted to the Central Bank of Nigeria (CBN).
But Mr Sanusi still says that $20bn is missing.
"Out of the $67bn that has accrued to the NNPC account, we have accounted for $47bn," Nigeria's ThisDay newspaper quotes Mr Sanusi as telling the committee, which is investigating the missing money.
But Andrew Yakubu, the NNPC's managing director, said these were "unsubstantiated claims" which showed "little understanding of the technicalities of the oil industry".
"CBN is not an auditing outfit. But what it is doing now is auditing. We have no problem with auditing, but let the professionals, the certified bodies and agencies that are charged with this responsibility of auditing, do their work,'' he said in a statement.
A respected banker, Mr Sanusi has spearheaded reforms in Nigeria's troubled banking sector since his appointment in 2009.
In December, Mr Jonathan, who is facing criticism over his leadership from within his own party, said Mr Sanusi's allegations were "spurious".
In an open letter to his critics, the president also denied allegations of "high corruption" in government.
Last month, a source told the BBC that Mr Jonathan had demanded Mr Sanusi's resignation over the leaked memo, but the central bank chief refused to go.
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