Chitosan and its derivatives have a wide range of applications in the field of enzyme immobilization. Understanding the mechanism of the interaction between enzymes and chitosan derivatives at the molecular level is of great significance to the design and application of related biocatalysts. In this study, molecular dynamics simulation was used to investigate the interaction of Candida Antarctica Lipase B (CaLB) and oligo-chitosan (OCTS), and molecular docking of lipase and catalytic substrate was conducted to study the binding conformation and affinity for the catalytically active centre of CaLB and substrate. The results showed that both electrostatic and L-J interactions between CaLB and OCTS play important roles in the initial stage of the self-assembly process. After the simulation process, the CaLB-OCTS assembly contained 14 OCTS molecules and 1 CaLB molecule. The electrostatic interaction and hydrogen bond interaction existed between CaLB and OCTS. During the simulation, the L-J potential energy and Coulomb potential energy decreased by about 1480 KJ/mol and 2324.0 KJ/mol, respectively, with the average number of hydrogen bonds between CaLB and OCTS increasing from 0 to about 17, and the accessible area of both CaLB and OCTS reducing by 20 nm2 due to the interactions. The analysis of secondary structure showed that compared to the free enzyme, the beta-sheet content of CaLB-OCTS increased from 9.78% to 12.62%, and the content of random coil loop decreased from 49.21% to 46.50%. Moreover, the CaLB-OCTS retained its original protein skeleton structure and the conformation of the active site pocket.