The state government says the dense gathering of cuttlefish off Whyalla is the only one of its kind in the world and is also a major tourism drawcard for the town.
The ban covers all waters north of Arno Bay on the gulf's west coast and Wallaroo on the east coast.
Primary Industries and Regional Development Minister Clare Scriven said the ban delivered on a Labor election promise.
"The annual migration of Australian giant cuttlefish to the waters of the upper Spencer Gulf to breed is one of the most spectacular natural events in the Australian marine environment," she said.
"It's right here on Whyalla's doorstep and people from around the world come to make the most of this incredible experience."
Ms Scriven said the protection order ensured a balance between sustainability, regional tourism benefits and the taking of aquatic resources in the area.
It does not prevent recreational and commercial fishers targeting other fish species including squid and octopus.
Local MP Eddie Hughes said the protection order would provide extra insurance for the species, particularly when cuttlefish were moving to the spawning grounds, and during the spawning season.
"The closure protects and preserves one of the natural wonders of South Australian marine life," he said.
Sometimes called the chameleons of the sea, cuttlefish can grow to 60cm in length and weigh up to five kilograms.
They go to the Spencer Gulf each year to spawn, with tens of thousands arriving between May and August to mate and reproduce.
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