The invention discloses a detection reagent for N-acetyl-beta-D-glucosidase. The detection reagent comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises 10-100mmol/L of citric acidbuffer solution, 0.1-0.3g/L of substrate, 1KU/L-5KU/L of ascorbic acid oxidase, 0.01-0.03g/L of magnesium chloride, 1-10g/L of stabilizer, and 2g/L-5g/L of preservative, the reagent R2 comprises 10-100mmol/L of sodium carbonate buffer solution, 3mL/L-5mL/L of triton X-100, and 2g/L-5g/L of preservative, the citric acid buffer solution is at 25 DEG C and the pH thereof is 4-5, the substrate is 5-[4-(3-methoxy-benzophene-rhodanine)-3-ammonium acetate-N-acetylamino-beta-D-glucoside, the stabilizer is one of EDTA, EDTA-2Na, glycol, and diglycolamidic acid, and the preservative is one of NaN3, methylparaben, and 4-chloro-3,5-dimethylphenol. A synthesized substrate 5-(4-(3-methyl-styrene)-rhodane-3-acetic acid amino-N-acetamino-beta-D-glucoside is used as a reaction substrate, the substrate isrelatively low-cost, and a reactant has high color development performance, so that the reagent is more sensitive.