Three men broke into the house of Kent Carpenter, 73, in the coastal town of Sibulan in Negros Oriental province during a suspected robbery on Sunday.
One of them is alleged to have shot the scientist in the head with a handgun as he sat on a sofa, police officials said.
Another suspect forced Carpenter's Filipino companion into a room and raped her.
The suspects took a laptop, an unspecified amount of cash and a backpack before fleeing, police spokesman Colonel Allen Rae Co said.
The third man is being hunted, Co told a news conference, adding that investigators were trying to determine why the suspects shot Carpenter.
A fourth suspect, who acted as a lookout outside the property, was arrested after surrendering to police and allegedly provided crucial details of the crime.
One of those arrested had previously been hired by Carpenter to do some carpentry work in his house, Co said, adding that the arrested suspects include the alleged gunman.
"All indicators as of now point to the fact that the attack was not connected to (Carpenter's) work," Co told reporters.
"It was really a planned robbery. So, we are still verifying why they killed the American marine biologist."
Several US and Philippine environment and biodiversity centres and universities have expressed shock over Carpenter's violent death.
Carpenter had testified for the Philippine government when it took China to international arbitration over longstanding disputes in the South China Sea.
His testimony as a biodiversity expert centred on the environmental effects of China's island-building and fishing activities in the disputed waters, according to former Philippine officials who had knowledge of his contribution.
The arbitration panel in The Hague invalidated China's expansive claims and pointed out the environmental damage caused by China's construction of islands in the disputed waters in a July 2016 decision.
China, however, questioned the panel's jurisdiction, refused to participate in the arbitration and rejected its ruling as a sham.
Carpenter had been a longtime lecturer and researcher at the Silliman University in Negros Oriental.
He had also been a biological sciences professor at Old Dominion University in Norfolk, Virginia, since 1996.
His research - which focused on the Philippines and the Coral Triangle between the Indian and Pacific oceans - shaped conservation efforts around the world, officials of the US university said.
They said he was on an extended research assignment in the Philippines and planned to retire in September.
"He dedicated his career to expanding our understanding of the world's bodies of water and protecting some of its most vulnerable ecosystems," Old Dominion president Brian Hemphill said.
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