The blast was likely caused by an anti-theft system on the door of a safe that Jamal al-Jamal was opening at the time, Prague police said.
The 56-year-old suffered "very serious injuries" in the blast and was taken to Prague's military hospital in an artificial coma, said Jirina Ernestova, spokeswoman for the emergency services.
Police later confirmed he had died.
"The evidence the police has doesn't suggest anything like a terror attack or that a specific person would set up a system with the intention to hurt or kill anyone," police spokeswoman Andrea Zoulova told AFP.
Daniel Langer, surgeon at the Prague military hospital to which Jamal was taken, told Czech television the ambassador had suffered devastating "head, belly and chest injuries following an explosion".
Jamal, who took office in October, had only recently moved to the new residence on the northern outskirts of Prague.
Palestinian foreign minister Riyad al-Malki hailed him as "an exemplary diplomat, who served his country and cause well".
The Palestinian foreign ministry said the blast occurred on Wednesday morning as Jamal "was opening an old safe which had been brought from the previous embassy (building) to the new one.
"Minutes after opening the safe the explosion took place, causing serious injury to Jamal who was taken to hospital and operated on," the ministry said in a statement.
Zoulova said police were not ruling out the possibility the safe was "mishandled", adding "the victim has died so it will be harder to prove the cause".
Malki said no foul play was suspected, noting that the safe had been left untouched for more than 20 years.
The safe was recently moved from the old embassy building, but it had come from a building that used to house the Palestinian Liberation Organisation's offices in the 1980s, Malki said.
"The ambassador decided to open it. After he opened it, apparently something happened inside (the safe) and went off," Malki told The Associated Press.
"The safe was sitting neglected in one of the areas of the old
embassy. It was in one of the corners. No one had touched it for 20 to 25 years....
"The ambassador wanted to know what is in the safe. He opened it and asked his wife to bring a paper and a pen to write down the contents of the safe. She left him to bring (the) pen and paper.
During that time, she heard the sound of an explosion," Malki told AP.
Police searched a building next door, which also belongs to the Palestinian embassy, but said they had not found any other explosives in the area.
"Still, this (explosives) is a thing you shouldn't find at any embassy and we will continue a probe into this within the investigation," Czech police chief Martin Cervicek told the private Nova television station.
Embassy spokesman Nabil al-Fahel told Czech radio that the ambassador's entire family had been in the residence when the blast occurred.
Jamal's 52-year-old wife "was taken to another hospital because of smoke inhalation and a stress reaction", Ernestova told AFP. The wife was released from hospital later on Wednesday.
Fahel said a special Palestinian team had arrived in Prague and got the green light from the Czech foreign ministry "to take part in the investigation of today's incident".
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