A creatine amidinohydrolase having the following physicochemical properties: Action: catalyzing the following reaction; creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum temperature: about 40-50.degree. C. Optimum pH: pH about 8.0-9.0 Heat stability: not more than 50.degree. C. (pH 7.5, 30 min) Km value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase: about 3.5-10.00 mM Molecular weight: about 43,000 (SDS-PAGE) Isoelectric point: 3.5 4.5, a method for producing said enzyme, comprising culture of microorganism producing said enzyme, a method for the determination of creatine or creatinine in a sample using said enzyme, and a reagent therefor.