The invention discloses a preparation method and application of cheese rich in gamma-aminobutyric acid (GABA), belonging to the technical field of food processing. The method provided by the invention comprises the steps of respectively activating a main leavening agent and an auxiliary leavening agent, adding sodium glutamate and pyridoxal phosphate into raw milk, sterilizing, then cooling, adding the main leavening agent and the auxiliary leavening agent, carrying out constant-temperature leavening, adding CaCl2, then carrying out constant-temperature leavening again, adding chymosin for curding, then cutting, standing at room temperature, heating, stirring for clotting, carrying out heat-preservation stirring, adding salt, and carrying out ripening processing, wherein the auxiliary leavening agent is freeze-dried bacterial powder of Lb. plantarum NDC 75017. Through the method, the cheese rich in GABA can be prepared, the yield of GABA in the cheese reaches up to 108.34mg/100g, the addition of Lb. plantarum NDC 75017 cannot influence the physicochemical indexes, the microbiological indicators and the sensory characteristics of the cheese, meanwhile, the total content of free amino acid is obviously increased, the taste and the smell of the cheese can be improved, and the sensory evaluation of the cheese is obviously higher than that of a blank reference group.