This study aimed to investigate whether charged short peptides influence enzyme activity and structure in the interaction system between enzymes and substrates, and to explore the regulatory mechanism of synthetic charged short peptides on α-amylase. In this study, the synthetic charged short peptides T1+ and T2– were introduced into the α-amylase catalytic system, and key indicators such as activity of α-amylase and zeta potential were measured. The results showed that the addition of short peptide T1+ reduced the activity of α-amylase by 1.29%, and the addition of short peptide T2– increased it by 0.4%. Both peptides induced structural changes in the enzyme. The results of molecular docking showed that the conformational interaction between α-amylase and substrate was changed by the charged short peptide. Based on these findings, it is hypothesized that the regulatory mechanism of synthetic charged short peptides involves modifying the enzyme's surface structure and active site, thereby influencing its catalytic activity.