Abstract: Objective To investigate the effect of antibodies against DNA binding protein HU on the biofilm formation of Staphylococcus aureus (S.arueus). Methods The gene HU was amplified by PCR using S.aureus NCTC-8325 genome DNA as template, and the recombinant vector pET-28a-HU was constructed and transformed into Escherichia coli BL21 (DE3). The expression of HU protein was induced by IPTG and purified by Ni²⁺ affinity chromatography. The polyclonal antibodies against HU protein (anti-HU) were prepared and purified by Protein A affinity resin. The interaction between HU and anti-HU was measured by ELISA and the function of anti-HU was verified by the biofilm formation assay of S.aureus. Results Recombinant protein HU was expressed in soluble form, and a high titer (> 1∶100 000) rabbit anti-HU polyclonal antibody was successfully prepared.The results of crystal violet staining after biofilm formation showed that the absorbance of anti-HU IgG at OD595nm were 0.052 and 1.220 at concentrations of 75 mg/ml and 0.6 mg/ml, respectively. Confocal assay showed that anti-HU polyclonal antibodies significantly inhibited S. aureus biofilm formation in a concentration-dependent manner. Conclusion Our results present that the biofilm formation of S.aureus is significantly blocked by the specific antibodies against HU portien, which provides a novel alternative strategy for treatment of S.aureus infection.