Low-energy N⁺ implantation mutagenesis allows Staphylococcus aureus to develop resistance to vancomycin. Then the vancomycin-resistant S. aureus strain was examined using drug-resistance phenotypes, and biofilm growth analysis to explore the potential resistance mechanism underlying the vancomycin-resistant S. aureus strain. The results showed that 9 vancomycin-resistant S. aureus strains were constructed by low energy N⁺ implantation mutagenesis. The biofilm biomass and density as well as the matrix protein content within the biofilms of five representative vancomycin-resistant strains with muti-drug resistance capacity were significantly enhanced over the original strain, and their resistance to vancomycin increased by between two and four times. These vancomycin-resistant strains acquired multiple resistance to different antibiotics. The results indicate that the enhancement of biofilm formation ability and the matrix protein content within the biofilm might be attributed to be the changes in the expression of drug-resistant-related genes in vancomycin-resistant S. aureus strains, which were mediated by low energy N⁺ implantation, thereby accelerating the formation of drug resistance. These results provide the theoretical basis for examining the mechanism of environmental radiation-mediating the drug-resistant formation in S. aureus.