The invention discloses a preparation method of cheddar cheese rich in gamma-aminobutyric acid. The cheddar cheese comprises the following raw materials of raw milk, chymosin, calcium chloride, a fermenting agent and the like. The cheddar cheese rich in gamma-aminobutyric acid is made through the working procedures of standardization of the raw milk, sterilization, cooling, innoculating streptococcus thermophilus and a commercial direct-vat type fermenting agent, generating acid, adding the calcium chloride, adding the chymosin for obtaining milk curds, cutting the milk curds, cheddaring, crushing the milk curds, salting, and maturing. The gamma-aminobutyric acid has the effects of regulating the blood pressure, stimulating the insulin secretion, restraining arteriosclerosis, improving the functions of the liver and the kidney, activating cerebral metabolism, calming and the like. According to the preparation method disclosed by the invention, the streptococcus thermophilus and the commercial direct-vat type fermenting agent are compounded, so that the content of the gamma-aminobutyric acid is continuously accumulated in the making and maturing stage of the cheese, and the content of the gamma-aminobutyric acid in the cheddar cheese is 10-100mg/100g. The cheese not only reserves the nutrient value, but also increases the physiology efficacies of the gamma-aminobutyric acid, so that the added value of products is increased.