Canadian activists say the ships, which made a failed attempt to challenge the blockade last summer, left Turkey on Wednesday and could reach Gaza in a couple of days.
Dylan Penner, a spokesman for the Canadian boat, the Tahrir, said there are about a dozen peace activists and journalists on board, including three Canadians.
Penner said Ottawa and other governments around the world should demand that Israel does not attack the unarmed civilians on the boats.
Ottawa issued an advisory warning Canadians against all travel to Gaza, saying the security situation along the coast remains volatile.
Foreign Affairs said the Canadian government can not offer protection to Canadians who break the laws of another country.
"I strongly urge those wishing to deliver humanitarian goods to the Gaza Strip to do so through established channels," Foreign Affairs Minister John Baird said in a statement.
"Unauthorized efforts to deliver aid are provocative and, ultimately, unhelpful to the people of Gaza."
Baird also recognized Israel’s right to protect itself and its people from attacks by Hamas and other terrorist groups, including by preventing the smuggling of weapons.
An Israeli army spokeswoman said the military knew about the boats and the navy is prepared to intercept them.
"This is a provocation in a long line of provocations," said Lt. Col. Avital Leibovich, adding that the activists were "more than welcome" to send aid to Gaza by land.
A similar flotilla last year ended in bloodshed — nine Turkish pro-Palestinian activists were killed and 45 injured after Israeli soldiers boarded a Turkish vessel trying to break the blockade.
Israel imposed a sea blockade on Gaza in 2007 after Hamas militants seized control there.