In this work, a chitosan/alginate multilayer film was constructed via layer-by-layer self-assembly and studied by in situ surface plasmon resonance monitoring and contact angle measurements. The results demonstrate that the surface composition of the self-assembled multilayer film can be simply tailored through pH control during the assembly process. The biological property of the assembled film was further characterized via antigen-antibody interactions, showing that the loading capacity of the antibody on the multilayered film and the binding activity of the antigen to the immobilized antibody could be also well-tuned by pH control. This work can provide more scientific insight in the interaction between protein and polymer matrix and render a novel simple approach to build high-performance biointerfaces through pH control for potential applications of highly sensitive immunosensors.