The application of biotechnological products in the feed industry has undergone explosive growth in recent years, and phytase from microorganism accounts for one-third of the entire feed enzyme market. In this study, some differences in the composition of protein and denaturation temperature between two commercial phytases were determined by HPLC and differential scanning calorimetry, which were derived from the same origin of E. coli. At the same time, we found that it was advantageous for near-infrared reflectance spectroscopy (NIRS) to display the protein differences in the commercial phytase, which is most important for ensuring the traceability of biotechnological products in feed and food safety control. Furthermore, NIRS could track the changes in phytase during the spray-drying process and the change of enzyme activity during storage of phytase. Our experiments proved that the information from NIRS could describe well the individual characteristics of the commercial phytase, which indicated that near-infrared reflectance spectra could be exploited to use in the registration system of commercial phytase.