Driven by the serious consequences caused by biofouling, fouling-resistant coatings integrated ‘offense’ and ‘defense’ properties are developed for combating marine biofouling. In this work, polysaccharides (PSa)-based self-polishing multilayer coatings were prepared via layer-by-layer (LbL) deposition. Dextran aldehyde (Dex-CHO) and carboxymethyl chitosan (CMCS) were synthesized and alternatively incorporated via imine linkage into the multilayer coating. X-ray photoelectron spectroscopy (XPS), surface plasmon resonance (SPR) and ellipsometric thickness profiles were utilized to characterize the LbL assembly. Experimental observes showed that the antifouling and antimicrobial performances of the resulting multilayer coating against bovine serum albumin (BSA) adsorption, bacterial adhesion (S. aureus and E. coli), and Amphora coffeaeformis attachment improved steadily, with increasing number of assembled bilayers. More importantly, the pH-responsive imine linkages-triggered self-polishing ability was achieved via bond cleavage under acidic environments. Besides, dense bacterial adhesion also induced detachment of the outmost layer of the multilayer coating. The anti-adhesion and antimicrobial efficacies were thus enhanced by the self-detachable capability of the coating layers. Therefore, the LbL-deposited self-polishing dextran/chitosan coatings offer an environmentally friendly and sustainable alternative for biofouling inhibition in aqueous and marine environments.