Staphylococcus aureus has the potential to opportunistically cause infectious diseases. The aim of this study was to determine the antimicrobial effects of novel graphene oxide (GO)-polyethylenimine (PEI)-based antisense yycG (ASyycG) on the inhibition of methicillin-resistant S. aureus (MRSA) biofilm formation. In current study, a novel GO-PEI-based recombinant ASyycG vector transformation strategy was developed to produce ASyycG. The mechanical features including zeta-potential and particle size distributions were evaluated by: GO; GO-PEI and GO-PEI-ASyycG. The recombinant ASyycG vector was transformed into MRSA cells, and the expression levels of the yycF/G and icaADB genes were determined and compared by quantitative real-time PCR (qPCR) assays. The recombinant ASyycG plasmids were subsequently modified with a gene encoding enhanced green fluorescent protein (ASyycG-eGFP) as a reporter gene, and the transformation efficiency was assessed by the fluorescence intensity. The biofilm biomass and bacterial viability of the MRSA strains were evaluated by crystal violet assay, colony-forming unit assays and confocal laser scanning microscopy. The results showed that the Z-average sizes of GO-PEI-ASyycG were much larger than those of GO or GO-PEI. The GO-PEI-based strategy significantly increased the efficiency of ASyycG transformation. The GO-PEI-ASyycG-transformed MRSA strain had the lowest expression levels of the biofilm formation-associated genes. Furthermore, GO-PEI-ASyycG suppressed biofilm aggregation and improved bactericidal effects on the MRSA after 24 hr of biofilm establishment. Our findings demonstrated that GO-PEI based antisense yycG RNA will be an effective method for management of MRSA infections.