Bacterial infections occur frequently during the implantation of medical devices, and functional coating is one of the effective means to prevent and remove biofilms. In this study, three different hydrophilic polyoxazolines with carboxyl groups (aPOx: PT1, PT2 and PT3) and bactericidal silver nanoparticles (AgNPs) were synthesized successfully, and an aPOx-AgNP multilayer film was prepared by electrostatic layer-by-layer self-assembly. The effect of charge density and assembly solution concentration was explored, and the optimal self-assembly parameters were established (PT2 1 mg/mL and AgNPs 3 mg/mL). The hydrophilicity of the surface can be enhanced to resist protein adhesion if the outermost layer is aPOx, and AgNPs can be loaded to kill bacteria, thereby realizing the bactericidal anti-adhesion potential integration of the aPOx-AgNP multilayer film. In addition, the aPOx-AgNP multilayer film was found to have the characteristic of intelligent and efficient pH-responsive silver release, which is expected to be used as a targeted anti-biofilm surface of implantable medical devices.