The invention discloses a creatinine detection reagent which is characterized by comprising a diluent and a reaction reagent, wherein the diluent is composed of buffer, surfactant, preservative, anti-bilirubin interference agent and vitamin C oxidase; and the reaction reagent is composed of buffer, creatinase, sarcosine oxidase, peroxidase, N-ethyl-N-(3-propylsulfo)-3-methylaniline, creatininase, 4-aminoantipyrine, preservative and freeze-drying protective agent. The detection reagent disclosed by the invention has favorable sensitivity, accuracy, precision and linearity, and can completely satisfy the clinical examination requirements.