Tania Farah said the Delta Airlines official "yelled" at the four-time Olympic gold medallist and refused to believe he had a business class ticket.
However, other reports say the Farahs arrived late for first-class boarding.
A Delta Airlines spokesman said it was investigating and "will be working directly with the Farah family".
The incident is alleged to have happened on 22 August as the couple and their four children were returning from the Rio Olympics, where Farah retained his 5,000m and 10,000m titles.
Farah and his family were on the final leg of their journey from Atlanta to their home in Portland, Oregon.
"I just knew she had a problem with him," Tania Farah said in the Sunday Telegraph, referring to the Delta Airlines official.
"This woman basically humiliated him until people came forward and said: 'That's Mo Farah, the Olympic champion'," his wife said.
"She was mortified afterwards, but had basically yelled at him.
"He was the only black person and hadn't done anything to warrant it."
Farah, 33, is aiming to become the first man to win three consecutive Great North Run titles when he races in Newcastle on Sunday.
Mo Farah was humiliated and sent to the back of the queue by an airline attendant before a flight in the United States, his wife has said.