The gunfire erupted during a 7 p.m. CDT (0000 GMT) showing of the film "Trainwreck" and took place almost three years to the day after a massacre at a cinema in Aurora, Colorado, that killed 12 people.
Lafayette Police Chief Jim Craft said two people died in the hail of bullets before the 59-year-old gunman killed himself with a handgun as officers responded to the scene shortly after 7:30 p.m.
Seven people suffered injuries ranging from non life-threatening to critical, Craft said. Authorities described the gunman, John R. Houser, as a drifter from Alabama who staying at a local motel.
Before purchasing a ticket for the show, Houser parked his blue Lincoln Continental, having switched its license plates, near the theater's exit and stashed a set of keys on the tire in what police said appeared to be preparations for a quick getaway. Disguises including wigs and glasses were later uncovered in his motel room.
"It is apparent that he was intent on shooting and escaping," Lafayette Police Chief Jim Craft said.
Houser, who police said came from Phenix City, Alabama, never made it back to his car, instead shooting himself as police swarmed the Grand 16 Theater, a multiplex located along main thoroughfare in Lafayette.
But investigators offered no immediate motive and the gunman's body remained inside the theater several hours later.
Witness Katie Domingue told the local Advertiser newspaper that the gunman stood up in the theater and began shooting about 20 minutes into the movie.
"He wasn't saying anything. I didn't hear anybody screaming either," Domingue said.
Keifer Sanders told CNN that some 100 people were in the theater when the gunman opened fire.
Police said they did not know why the suspect launched the attack in Lafayette, roughly 55 miles (90 km) southwest of Baton Rouge.
"Certainly it exists out there that we may not find a motive but that's not our goal right now," Colonel Mike Edmonson of the Louisiana State Police told reporters.
Republican Louisiana Governor Bobby Jindal traveled to Lafayette in the wake of the shooting.
"As governor, as a father and as a husband, whenever we hear about these senseless acts of violence it makes us both furious and sad at the same time," he said at a briefing.
The shooting came three years after a gunman opened fire at a movie theatre in Aurora, Colorado, during a screening of the Batman film, "The Dark Knight Rises", killing 12 people and wounding 70 others.
Former graduate student James Holmes was convicted last week on 165 counts of murder, attempted murder and explosives in the July 20, 2012, rampage.
Jurors in that case were trying to determine if Holmes should face the death penalty or life in prison during a penalty phase of that case.
The United States has witnessed several mass shootings in the last two months.
A gunman is accused of a racially motivated shooting at a black church in South Carolina that killed nine church members in June. More recently, a gunman attacked military offices in Tennessee last week, killing five U.S. servicemen.
Jindal said he had ordered National Guard members at offices and other facilities to be armed in the wake of the Tennessee attack.
In a BBC interview excerpt that aired on Thursday before the shooting, U.S. President Barack Obama said his biggest frustration was the failure to pass "common-sense gun safety laws" in the United States.