Lieutenant-Colonel Samir Shehade and three other men were wounded when an explosion ripped through their convoy as it left the town of Rmeileh, about 30km south of Beirut early on Tuesday.
Ahmed Fatfat, the Lebanese interior minister, said the blast was caused by a roadside bomb loaded with nails.
The attack came 10 days ahead of the release of a report on al-Hariri's assassination, which Western countries suspect was the work of Syria.
A massive blast killed al-Hariri and 22 other people on February 14 last year.
In September last year, Shehade, the deputy director of the internal security force's intelligence unit, directed the arrest of four top Lebanese intelligence officials believed to have been involved in al-Hariri's murder.
Lebanese security officials, speaking on condition of anonymity because of the sensitivity of the case, said Shehade had received threats because of his work in the al-Hariri probe.
Al-Hariri's son, Saad Hariri, a prominent lawmaker in Lebanon, called the attack a terrorist act.
"This is a message which we reject," he said.
Fares Soueid, a former parliamentarian and Saad Hariri ally, said the attack was aimed at sabotaging the creation of an international tribunal over al-Hariri's assassination.
Syria has denied any responsibility for the killings, but has come under heavy international pressure to co-operate with the investigations.
An international outcry over the murder of al-Hariri forced Syria, which had dominated Lebanese politics for three decades, to withdraw its troops from Lebanon in April 2005.
Since then, relations between the two neighbouring countries have soured considerably, and leaders of the anti-Syrian majority that emerged in parliamentary elections two months later have repeatedly claimed they fear for their lives.