A spokesman for the Israeli army confirmed that Aziz Dweik, 58, was arrested at his home on Saturday night.
"We arrested him because he is a leader of Hamas, which is a terrorist organisation," said the spokesman.
Sixty-four other leading members of the Islamist movement, including eight ministers and 26 other politicians were also detained on June 29.
They were grabbed as part of a massive Israeli sweep against Hamas following the capture of an Israeli soldier by fighters on the border of the Gaza Strip on June 25.
Dweik's wife said that Israeli troops also seized two computers and documents of the Palestinian parliament when they arrested her husband.
Saeb Erikat, the Palestinian chief negotiator, condemned the arrest and demanded that Dweik and the other detainees be released.
Dweik was elected speaker in February.
He had previously been arrested five times by Israeli authorities, and was also part of a group of 400 Palestinians who were banned by Israel in 1992 and exiled to south Lebanon.
Elsewhere, Israel continued its military operations in the southern Gaza Strip where an air strike killed a Palestinian early on Sunday, medics said.
Earlier on Saturday, five more Palestinians, including two fighters, were killed in the Rafah area by Israeli missiles.
Israeli forces moved into the southern region on Thursday to destroy what the army called "terrorist infrastructure".
Medics said the Palestinian killed in the latest air raid was a civilian and that four others were wounded.
The Israeli army had no immediate comment.
Israel's Gaza offensive has killed at least 167 Palestinians, more than half of them civilians.