Buddhist monks and Brahmin priests swore in Cambodian and foreign judges in a ceremony at the Royal Palace on Monday.
Reach Sambath, tribunal spokesman, said: "It will end negative speculation that the trials will not take place."
Twenty-seven legal experts - 17 Cambodians and 10 foreigners - have been appointed to the tribunal, which has a three-year budget of $56.3 million.
Helen Jarvis, the tribunal's Australian chief spokeswoman, said: "It is a very historic day, something that people have been waiting for so many years.
"Now we are really starting the judicial process."
Cambodians had feared that they would never have their questions about the Khmer Rouge answered. Almost every family lost relatives under the 1975-79 regime, and none of its top leaders, some of whom are alive and living quietly in Cambodia, has faced trial.
The setup of the tribunal was worked out through years of negotiations with the United Nations.
No date has been set, but trials could begin early next year after prosecutors have assembled their cases.
Pol Pot, the "Brother Number One" and leader of the Khmer Rouge, died in 1998 in his jungle hideout nearly 10 years after a Vietnamese invasion destroyed the regime's attempt to create an agrarian utopia.
Nuon Chea, "Brother Number Two", Khieu Samphan, former head of state, and Ieng Sary, former foreign minister, are all living in the northwest near the Thai border.
About 1.7 million people died
under the Khmer Rouge
Under the Khmer Rouge, an estimated 1.7 million people were killed - some with a blow to the head with a hoe to save bullets - or died of overwork, starvation and disease.
Religion, property rights, currency and schools were all abolished as the regime turned the country into a collective farm.
Only two top officials are in custody, accused of war crimes, genocide and crimes against humanity.
Ta Mok, the one-legged Khmer Rouge military chief who is now 82, said last week after being taken from his jail cell to hospital that he wanted a swift trial.
His lawyer, Benson Samay, said Ta Mok appeared to fear he would not get to tell his side of the story before he died.
The other detained Khmer Rouge official is Duch, now a born-again Christian. He ran the Tuol Sleng interrogation centre which few prisoners survived.
One Cambodian and one international prosecutor will begin their investigation into the genocide. Prosecutors will decide which of the surviving leaders should face trial.