The interactions between nicosulfuron and two degradation enzymes (vegetative catalase 1 and manganese ABC transporter) from the Bacillus subtilis YB1 strain were studied and molecular docking simulations and surface plasmon resonance (SPR) were used to research their specific interaction patterns and affinities. The results showed that vegetative catalase 1 and manganese ABC transporter bound specifically to nicosulfuron and that the former binding ability was stronger than that of the latter. The manganese ABC transporter mainly interacted with nicosulfuron by strong hydrophobic interactions and hydrogen bonds (oxyanion hole), while vegetative catalase 1 formed a strong hydrophobic interaction with nicosulfuron in its main channel and hydrogen bond with nicosulfuron in the side chains. Vegetative catalase 1 and manganese ABC transporter catalyze nicosulfuron degradation, and molecular docking simulations and SPR are good methods for studying molecular interactions, which could make a foundation for the study of degradation mechanisms of enzymes.