The invention discloses a creatine kinase isoenzyme detection reagent. The reagent consists of a reagent R1 and a reagent R2 according to the volume ratio of 4 to 1, wherein the reagent R1 consists of an imidazole buffer liquid, glucose, nano particles, N-acetylcysteine, sodium ethylene diamine tetracetate, adenosine diphosphate, cozymase I, ribonucleotide, pyruvate decarboxylase, glucose 6-phosphate dehydrogenase, hexokinase, a goat anti-human CK-M polyclonal antibody and trehalose; the reagent R2 consists of an imidazole buffer liquid, phosphocreatine, sodium dodecyl benzene sulfonate and a preservative. The reagent disclosed by the invention is a creatine kinase isoenzyme detection reagent which is stable, accurate, and high in anti-interference, and has very high clinical application value.