The invention provides a glutathione reductase determination kit, and a preparation method and applications thereof, and relates to the technical field of biochemical reagent determination. The kit includes a reagent R1 and a reagent R2; the reagent R1 includes a phosphate buffer solution, ascorbic acid oxidase, pyruvate oxidase, potassium ferrocyanide, oxidized glutathione, MgCl2, FAD, a preservative and a surfactant; and the reagent R2 includes a glycine buffer solution, NADPH, the preservative and a protective agent. Through the adding of EGTA and dithiothreitol in the reagents, the linearrange of the kit can be enlarged, and the stability of the kit can be enhanced. The preparation method and applications of the kit are also provided; and the kit can accurately determine glutathione reductase.