The Pontiff led thousands of people in prayer for the 29 victims, who he said were killed in “another act of ferocious violence” after having refused to renounce their Christian faith at gunpoint.
Speaking from his studio window over St Peter's Square, Francis said: “May the Lord welcome these courageous witnesses, these martyrs, in his peace and convert the hearts of the violent ones.”
Isis claimed responsibility for the attack on Christians travelling through the Minya region to the St Samuel the Confessor monastery.
The bus was blocked by three vehicles carrying eight to 10 attackers who opened fire, killing at least 29 and wounding 24, with many children among the victims.
Footage showed the bus raked with bullets, with its windows smashed, surrounded by dead bodies covered in black plastic sheets.
It was the fourth attack against Christians in Egypt claimed by Isis since December. The string of attacks have killed more than 100 and injured scores.
Video interviews with survivors painted a picture of untold horror as children hid under their seats to escape the gunfire.
One survivor, a small boy who seemed to be about six, said his mother pushed him under her seat and covered him with a bag.
A young woman speaking from her hospital bed said the assailants ordered the women to surrender their jewellery and money before they opened fire, killing the men first and then some of the women.
The woman said the gunmen were masked and wore military uniforms.
Bishop Makarios, the top Coptic Orthodox cleric in Minya, the province where the attack took place, said the assailants told Christian men they ordered off the bus they would spare their lives if they converted to Islam.
“They chose death,” said Mr Makarios, who has been an outspoken critic of the government's handling of anti-Christian violence in Minya, where Christians account for more than 35 per cent of the population, the highest anywhere in Egypt.
“We take pride to die while holding on to our faith,” he said.
Mr Makarios confirmed the assailants stole the women's jewellery and his contention the men were ordered off the bus before being killed was also confirmed by a video clip purportedly taken in the immediate aftermath of the shooting.
The video showed at least four or five bodies of adult men lying on the desert sand next to the bus; women and other men screamed and cried as they stood or squatted next to the bodies.
The Coptic church said it had received news of the killing of its “martyrs” with pain and sorrow.
Egypt responded to the attack with a wave of air strikes against suspected militant bases where the military said the perpetrators trained.
A manhunt for the assailants in the vast deserts to the west of the site of the attack has so far yielded no arrests.
On Saturday, the Pope said there are more Christian martyrs today than in ancient times.
During a meeting with clergy in the Italian port city of Genoa, Francis urged them to pray “for our brothers the Egyptian Copts, who were killed because they did not want to renounce their faith”.
“Let’s not forget that today there are more Christian martyrs than in ancient times, than in the early day times of the church,” he told bishops, priests and nuns gathered in the Cathedral of San Lorenzo.
He has previously paid tribute to Christians in the Middle East who have been persecuted by Islamist militants and he has often denounced Isis and condemned the concept of killing in God's name.
The attack is the second time Isis has targeted the beleaguered Christian minority in Egypt in recent months following twin bombings that killed at least 44 people in churches in Tanta and Alexandria on Palm Sunday last month.
It has also particularly persecuted the Yazidi communities in Iraq – men were murdered and women were sold into sex slavery when the group declared its “caliphate” in 2014.
Additional reporting by agencies
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