The gunmen in three pick-up trucks attacked the bus as it carried visitors to the Saint Samuel monastery in Minya province.
The gunmen in three pick-up trucks attacked the bus as it carried visitors to the Saint Samuel monastery in Minya province, more than 200 kilometres (120 miles) from Cairo, before fleeing, the interior ministry said.
It was the latest attack on Copts after Islamic State group jihadists bombed three churches in December and April, killing dozens of Christians.
Pictures of the bus aired by state television showed its windows shot out.
"They used automatic weapons," Minya governor Essam el-Bedawi told state television.
Bedawi said police were fanning out along the road where the attack took place and had set up checkpoints.
Health ministry spokesman Khaled Megahed that 26 people were killed and another 25 wounded.
The shooting followed church bombings in December and April claimed by the Islamic State (IS) group.
The jihadists have threatened more attacks against the Copts, who make up about 10 percent of Egypt's 90-million population.
Suicide bombers with the jihadist group struck a Cairo church on December 11, next to the seat of the Coptic pope, killing 29 people.
On April 11, bombers attacked two churches north of Cairo on Palm Sunday, killing 45 people, in the deadliest strike in living memory against the Copts.
The Copts' Pope Tawadros II had been leading a service in one of the two churches attacked that day.
The bombings prompted President Abdel Fattah al-Sisi to declare a three-month state of emergency.
The Egyptian affiliate of IS has also killed several Copts in North Sinai, forcing dozens of families to flee the province in January.
Friday's shooting came after a historic visit to Egypt by Roman Catholic Pope Francis to show solidarity with the country's Christians.
In his late April trip, Francis visited one of the bombed Coptic churches and condemned violence carried out in the name of God.
Al-Azhar, Egypt's top religious authority, condemned Friday's shooting which took place on the eve of the start of the Muslim holy month of Ramadan.
"The Minya incident is unacceptable to Muslims and Christians and it targets Egypt's stability," Al-Azha’s Grand Imam Ahmed al-Tayeb said in a statement.
Copts have suffered sectarian attacks for years.
A suicide bomber had struck a church in 2011, and there have been deadly clashes with Muslims, especially in the rural south, following disputes over church construction.
Egypt says it has identified those behind the church bombings in April, saying they were part of an extremist cell based in southern provinces, offering a reward for their capture.
Sisi has defended the performance of his security forces and accused jihadists of trying to divide Egyptian society by attacking vulnerable minorities.