OBJECTIVE To establish a more sensitive, simple, accurate and stable method for determining the activity of recombinant human α-galactosidase A (rhα-GAL) based on the kinetic theory of enzyme reaction and study the assay conditions for the activity assay. METHODS The optimum conditions of the assay system were as follows: 50 mmol·L^-1 p-nitrophenyl-α-D-galactopyranoside was used as the substrate, the concentration of the enzyme was 1.67 μg·mL^-1, the reaction was accurately carried out in a water bath at 37 ℃ for 15 min, then the reaction was terminated by glycine buffer (pH 10.5), and the absorbance was measured at 400 nm using a microplate reader. RESULTS The method had good specificity. Rhα-GAL showed a good linear relationship with the enzymatic reaction rate in the range of 0.83-2.51 mg·mL^-1 (r=0.999 8). The recoveries of validation solutions at 50%, 80%, 100%, 125% and 150% concentrations were in the range of 94.2%-101.8% (n=18), and the CVs of the measured results were between 2.0% and 5.5% (n=18). The CV of 12 independent tests of the same sample was 2.21% (n=12). The effects of slight changes in water bath temperature, reaction time and substrate concentration in the reaction system on the results were investigated,confirming the good robustness of the method. The reconstituted sample showed good stability when stored at 2-8 ℃ for 48 h. p-Nitrophenol showed a good linear relationship with the absorbance in the range of 0.01-0.15 mmol·L^-1 (r=0.999 7). The recoveries of p-nitrophenol solution at five concentrations were in the range of 94.9%-105.1% (n=9),and the CVs were all below 2.0% (n=9). The activity of two rhα-GAL products was determined by this method. CONCLUSION A chromogenic substrate method was established to determine the activity of rhα-GAL and validated with good sensitivity, precision and accuracy, which can be used for the activity evaluation and quality control of the product.