The government of President Mahinda Rajapakse formally rejected rights chief Navi Pillay's demand for an external investigation into "credible allegations" that 40,000 ethnic Tamil civilians were killed by Sri Lankan forces in 2009.
Pillay's recommendation to a UN Human Rights Council (UNHRC) meeting in Geneva next month "reflects bias and is tantamount to an unwarranted interference in the internal affairs of a sovereign state", Colombo said in a statement.
The United States has already said it will move a third censure motion against Sri Lanka at the UNHRC, buttressing the recommendations of Pillay who visited Sri Lanka on a week-long fact-finding mission in August.
Shortly before leaving the island, Pillay launched a scathing attack on Sri Lanka's failure to deliver on promises to ensure accountability and accused the Rajapakse regime of becoming "increasingly authoritarian".
Sri Lanka's latest criticism of the UN official, set out in a 23-page statement, accused her of having a "preconceived, politicised and prejudicial agenda which she has relentlessly pursued with regard to Sri Lanka".
Pillay is South African of Tamil origin.
In a UN document leaked 10 days ago but published officially on Monday, Pillay asked the UNHRC to set up an independent probe and said Colombo had "consistently failed to establish the truth".
Pillay's report noted fresh evidence of what took place during the final stages of the island's ethnic war that ended in May 2009 when an army onslaught crushed the Liberation Tigers of Tamil Eelam separatist group.
The UN has said up to 40,000 Tamil civilians may have been killed during the final months of fighting and blamed many of the atrocities on government forces, a charge Colombo vehemently denies.
A report released by the Public Interest Advocacy Centre in Australia earlier this month claimed that Sri Lankan soldiers committed the "vast majority" of crimes.
The UN has estimated that at least 100,000 people were killed during Sri Lanka's separatist war between 1972 and 2009.