As a new technology for food processing, plasma has good prospects for protein modification. This study investigated the effect of plasma on the activity of the α-amylase. The composition of the active particles in air plasma generated by spark discharge was analyzed and determined. Furthermore, the quantitative analysis of the active particles such as H2O2, O3, and -OH was made by the chemical detection method. Powdered α-amylase was treated with plasma in various conditions, in which α-amylase and the variation of α-amylase activity under the action of air plasma were quantitatively analyzed. The results showed that the concentration of active particles in the system was positively correlated with the action time for air plasma. After 5 min of plasma action, the concentration of O3 and H2O2 was large enough for food disinfection, but the concentration of -OH was smaller and its lifetime was extremely short. Moreover, it was determined that the optimum action time for the activation of solid powdered α-amylase by air plasma was 120 s. With higher energy, the air plasma acts directly on solid powdered α-amylase to destroy its spatial structure, resulting in enzyme inactivation, sterilization, and disinfection.