The bill was signed by Florida governor Charlie Crist on Friday and will become effective on July 1.
The Humane Society of the United States has welcomed the new law.
It was introduced to the Florida legislature, sponsored by Senator Victor Crist and Representative Luis Garcia, following horrific attacks on horses, who were butchered for their meat.
Their remains were found in pastures and rural roadsides centered around Miami-Dade County.
In all, more than 20 horses have lost their lives in little more than a year.
Police inquiries have so far resulted in four arrests in several isolated cases. Authorities say there is strong black-market demand for horse meat in southern Florida, which fuelled the market.
The string of killings prompted community meetings and the reward for the conviction of those responsible climbed to more than $US20,000.
The new law makes it a felony to kill, maim or mutilate a horse, and prohibits the transport, sale, distribution or possession of horse meat that is not acquired from a "licensed slaughterhouse".
Since no operations in Florida are licensed to slaughter horses, this amounts to an outright prohibition on the slaughter of horses for human consumption.
"The incidents of horses being stolen, killed, and butchered in Florida has shocked and outraged the people of this state," said Jennifer Hobgood, Florida state director for The Humane Society of the United States.
"This bill enshrines the special place that our equine companions occupy for all Floridians."
Representative Garcia said: "It is my hope that these harsh punishments will eliminate this horrendous crime in our state. You really can't understand the true limits of human depravity until you have spoken with someone who has had their pet butchered alive for meat; it is heart-breaking ..."
Senator Crist said, "This bill reflects the distaste that most Floridians have for the practice of killing and butchering them for human consumption."