The embodiment of the invention belongs to the field of medical examination and determination, and relates to a kit for determining activity of creatine kinase isoenzyme. The kit comprises a first reagent and a second reagent; the first reagent comprises a first buffer solution, adenosine diphosphate, disodium ethylene diamine tetraacetate, magnesium acetate, an activator, adenosine monophosphate, an anti-human CK-MM antibody, AP5A, glucose, NAD (P) + and a first preservative; and the second reagent comprises the following components: a second buffer solution, phosphocreatine, magnesium acetate, hexokinase, glucose-6-phosphate dehydrogenase, tetrazolium salt, diaphorase and a second preservative. The invention also relates to a determination method of creatine kinase isoenzyme activity. According to the technical scheme, the test sensitivity can be improved.