Nabil Hodhod, an ally of Mahmoud Abbas, the Palestinian president, and central Gaza commander of the powerful Preventive Security force, died on Wednesday when a blast ripped through his car. His deputy was wounded.
There was no claim of responsibility, but it comes at a time of an increasingly bloody power struggle between forces loyal to Abbas and the Hamas-led government.
Hodhod was the highest official to be killed in a week of violent incidents, sparked by Hamas's fielding of its own militia in defiance of a ban by Abbas.
Two other senior security officers were also targeted.
On Saturday, Tareq Abu Rajab, the intelligence chief, was seriously wounded in an explosion in his Gaza office, and on Sunday, Palestinian security forces found a huge bomb on the road used by security commander Rashid Abu Shbak to drive to his office.
Abu Shbak, a Fatah stalwart, has become a symbol of the conflict. Abbas appointed him to command three security forces nominally under the authority of the Hamas-controlled interior ministry.
Hamas responded by deploying its own 3,000-strong militia, setting off clashes with Fatah forces.