The invention relates to the field of an external diagnostic reagent, and particularly relates to a high-sensitivity kit for detecting 5'-nucleotidase. The kit for detecting 5'-nucleotidase comprises a 5'-nucleotidase R1 reagent, a 5'-nucleotidase R2 reagent and a 5'-nucleotidase calibration product, wherein the 5'-nucleotidase R1 reagent comprises a buffer solution, a stabilizing agent, a preservative agent, an enzyme activator, 4-AAP(4-aminoantipyrine), 18-crown ether-6 and UAO (Uricase), PNP (Purine Nucleoside Phosphorylase), XOD (Xanthine Oxidase) and POD (Peroxidase) reaction enzyme systems; the 5'-nucleotidase R2 reagent comprises a buffer solution, a stabilizing agent, a preservative agent, PO3- and a Trinder developing system; and the 5'-nucleotidase calibration product comprises a buffer solution, a stabilizing agent, a preservative agent and 5'-nucleotidase. The detection kit provided by the invention is enhanced in detection sensitivity by 40% compared with a 'PNP-XOD-POD' enzyme system, and has good application prospects.