The killing on Thursday came a day after the Tamil rebels and the government agreed to resume peace talks.
Seevaratnam Puleedevan, a top rebel peace negotiator, said the officer known as Kapilan had been "killed by paramilitary cadres with the backing of Sri Lankan armed forces."
However, the Sri Lankan military disavowed any involvement in the ambush.
"Our armed forces were not involved in any way," said military spokesman Brigadier Prasad Samarasinghe.
Kapilan was killed and a colleague wounded in an ambush as they rode on a tractor through a rebel-held area of eastern Sri Lanka, the pro-rebel TamilNet website said.
'Gesture of goodwill'
Commenting on the attack, Puleedevan questioned whether the government was serious when it agreed less than 24 hours earlier to restart peace negotiations that have been stalled since 2003
"We have taken the matter very seriously and have raised it with Mr. Solheim, [the Norwegian peace envoy] telling him to convey to the Sri Lankan government to immediately stop such provocative attacks. This attack shows the Sri Lankan government was playing a double game," Puleedevan said.
"Our leadership is very disturbed, as this is a very serious incident," Puleedevan said.
He said the Tigers recently released a Sri Lankan policeman held in their custody as a "gesture of goodwill."
For its part the Sri Lankan government condemned the killing, saying it still planned to go ahead with peace talks with Tamil Tiger rebels.
"This attack shows the Sri Lankan government was playing a double game"
Seevaratnam Puleedevan, Rebel peace negotiator
Anusha Palpita, the Director of the Government Information Department said: "The government is determined to go ahead and hold the talks, as agreed".
The government said it had received reports from rebel-held areas of "heavy fighting," but denied any involvement.
European ceasefire monitors in the area said they were investigating the attack.
The rebels in 1983 launched a violent campaign to create a separate state for ethnic minority Tamils in the northeast, accusing the majority Sinhalese of discrimination.
The civil war killed 65,000 people before the ceasefire was signed.