Abstract: Soil phosphatase, especially acid phosphatase, plays a critical role in the decomposition of organic phosphorus and has a major impact on plant phosphorus uptake. Most Chinese researchers refer to the book entitled Soil Enzyme and Its Research Method, edited by Songyin Guan, for measurement method of soil acid phosphatase activity based on phenyl phosphate disodium salt substrate. In contrast, researchers outside China mainly cite the book entitled Methods of Soil Enzymology, edited by Dick, that was based on disodium p-Nitrophenyl phosphate tetrahydrate (PNPP) substrate. However, non-conspicuous coloration has existed for the measurement of products based on phenyl phosphate disodium salt substrate. Furthermore, it has been difficult for researchers to select an optimal method for determining acid phosphatase activity since these methods use different substrates. To determine the optimal method for measuring soil acid phosphatase activity, three different methods were used to measure the acid phosphatase activity of 10 soil samples of acid, neutral and alkaline soils, respectively. The three selected methods were 1) based on phenyl phosphate disodium salt substrate and colored using pH 5.0 acetate buffer (DPP 1); 2) based on phenyl phosphate disodium salt substrate and colored using pH 9.4 borate buffer (DPP 2) during chromogenic process; or 3) the PNPP method. Furthermore, the study analyzed the effects of different pH buffers and phenol concentrations on product absorbance. The results showed that chromogenic reaction of phenol with 2,6-dibromchinone-chlorimide was colorless within pH° ≤° 6 buffer solution with phenyl phosphate disodium salt as the substrate. In contrast, the above chromogenic reaction was observed under alkaline buffer (pH°≥°8) in all the samples. And there were significant differences in the Pearson correlation coefficient (R²) between phenol concentration and product absorbance at 0.01 level. Therefore, pH was a significant factor in determining the coloration between phenol and 2,6-dibromchinone-chlorimide. Furthermore, when acid phosphatase activity was determined using the PNPP method, the coefficient of variation of acid phosphatase activities in the 10 soil samples increased by 70.04%, 42.44% and 21.17% in acid, neutral and alkaline soils, respectively, which was in sharp contrast to those determined using the DPP 2 method. The range of soil acid phosphatase activities determined by the PNPP method was 27.18, 26.85 and 39.43 times larger than those determined by the DPP 2 method in acid, neutral and alkaline soils, respectively. These results suggested that regardless of soil acidity, PNPP was an easier and more sensitive method than DPP 2 for the estimation of soil acid phosphatase activity. In addition, if phenyl phosphate disodium salt was used as substrate in an assay, alkaline borate was the most suitable buffer for coloration reaction systems.