Abstract: The application of chondroitinase requires consideration of the complex microenvironment of the target. Our previous research reported a marine-derived sodium dodecyl sulfate (SDS)-resistant chondroitinase VhChlABC. This study further investigated the mechanism of VhChlABC resistance to SDS. Focusing on the hydrophobic cluster on its strong hydrophilic surface, it was found that the reduction of hydrophobicity of surface residues Ala¹⁸¹, Met¹⁸², Met¹⁸³, Ala¹⁸⁴, Val¹⁸⁵, and Ile³⁰⁵ significantly reduced the SDS resistance and stability. Molecular dynamics (MD) simulation and molecular docking analysis showed that I305G had more conformational flexibility around residue 305 than wild type (WT), which was more conducive to SDS insertion and binding. The affinity of A181G, M182A, M183A, V185A and I305G to SDS was significantly higher than that of WT. In conclusion, the surface hydrophobic microenvironment composed of six residues was the structural basis for SDS resistance. This feature could prevent the binding of SDS and the destruction of hydrophobic packaging by increasing the rigid conformation of protein and reducing the binding force of SDS-protein. The study provides a new idea for the rational design of SDS-resistant proteins and may further promote chondroitinase research in the targeted therapy of lung diseases under the pressure of pulmonary surfactant.