Luis Moreno-Ocampo said no-one had the right to massacre civilians.
Thousands of people are thought to have died in the violence after security forces targeted protesters in unrest which began on 17 February.
Col Gaddafi vowed to fight on despite losing control of much of the country.
Earlier on Wednesday, his forces launched air strikes on the oil terminal town of Brega, sources in the town said.
The strikes come a day after clashes between rebels and government troops in the town in which 14 people died.
In other developments:
Libya's oil production has been halved, National Oil Corporation head Shukri Ghanem tells AFP
The Netherlands confirms three of its marines have been captured by pro-Gaddafi forces in Sirte while helping to evacuate two civilians
Libya's governing council rejects a peace initiative by Venezuelan President Hugo Chavez, but the Arab League says it is studying the proposal
'Serious crimes'
Mr Moreno-Ocampo said Col Gaddafi, his inner circle and some of his sons with de facto authority would be investigated.
"The office of the prosecutor decided to open an investigation into alleged crimes against humanity committed in Libya since 15 February," he said.
Efforts to evacuate the refugees are frustratingly slow "Delegations of peaceful demonstrators were attacked by security forces.
"During the coming weeks, the office will investigate who are the most responsible for the most serious incidents, for the most serious crimes committed in Libya."
But the prosecutor also said the oppposition would be investigated if it committed crimes.
The violence has led to a major humanitarian crisis on the Tunisian border, with tens of thousands of foreigners stranded and unable to get home.
Some 80-90,000 people have fled to Tunisia since the unrest began and tens of thousands more are waiting to cross, the UN says.
Until recently most of the refugees have been Egyptian, but more than 5,000 Bangladeshis crossed on Wednesday and there are also large numbers from East Asia.
The BBC's Jim Muir on the border says it will be a monumental task to get them home.
Britain, France, Spain and other countries are conducting emergency airlifts to evacuate those gathered at the border.
'Bombs dropped'
It was not clear what was being targeted in the air strikes on Brega.
"Around two hours ago, warplanes dropped a bomb in the area between the oil company and the residential area," hospital official Fattah al-Moghrabi said, quoted by AFP news agency.
"As far as I know, there were no casualties."
A rebel fighter said he had witnessed the strikes.
"I was here with my people, tightening security around the refinery, then we heard the sound of planes," he told AFP.
"It was one plane that flew over three times. I think it was a Sukhoi. The third time, it dropped bombs."
Rebels said planes had also bombed positions in Ajdabiya, a larger town further inside rebel territory.
The attacks came as rebels celebrated the outcome of Wednesday's fighting, in which they apparently cleared Brega of government forces.
BBC diplomatic correspondent Jonathan Marcus says the fighting underscores the fact that neither side seems to have the knock-out punch capable of defeating the other.
Col Gaddafi appears unable to re-capture the eastern part of his country, and for now his opponents seem unable to mount a major offensive against Tripoli.
A stalemate on the ground in Libya could be a serious problem for international diplomacy, our correspondent says.
Rebels were reported as saying pro-Gaddafi forces had been pushed back to Ras Lanuf, another oil terminal to the west of Brega, but were preparing another attack.
They are calling for UN-backed air strikes against pro-Gaddafi forces, but Western officials are cautious of military involvement.
Our correspondent says that diplomatic measures against Col Gaddafi were intended to hasten his departure and express support for the Libyan people's aspiration for freedom.
But there is now a danger that, having been painted into a corner, the Libyan leader may have little option but to cling on to power for as long as possible, he adds.
BBC
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