The biofilms could protect bacteria from antibiotics and promote the production of drug-resistant strains, making the bacteria more difficult to be eradicated. Thus, we developed an AMP@PDA@AgNPs nanocomposite, which is formed by modifying silver nanoparticles (AgNPs) with antimicrobial peptides (AMP) modified nanocomposite to destroy biofilm in this study. The AMP@PDA@AgNPs nanocomposite was prepared with polymerization method and characterized by using ultraviolet-visible (UV-vis) spectroscopy, dynamic light scattering (DLS), Fourier transform-infrared spectroscopy (FT-IR), and transmission electron microscope (TEM). The antibacterial effects of the nanocomposite were investigated by using agar diffusion method and minimum inhibitory concentration (MIC) test. The quantitative analysis of the biofilm formation by the nanocomposite was conducted using crystal violet staining and confocal laser scanning microscope (CLSM). The DLS and TEM analysis showed it was a spherical nanocomposite with 200 nm size and well dispersed . The results of UV-vis and FT-IR confirmed the presence of AMP and AgNPs. The nanocomposite had an excellent biocompatibility at 100 μg/mL. And the AMP@PDA@AgNPs nanocomposite showed superior antimicrobial activity against both Gram-negative (E. coli, P. aeruginosa) and Gram-positive (S. aureus) bacteria than AgNPs or AMP. Importantly, the mRNA expression of biofilm-related genes were decreased under the action of the nanocomposites. An AMP@PDA@AgNPs nanocomposite with good biocompatibility was successfully prepared. The nanocomposite could destruct bacterial biofilms by inhibiting the expression of biofilm-related genes. The synergistic strategy of AMPs and AgNPs could provide a new perspective for the treatment of bacterial infection.