Objective: To develop the gentamicin sulfate/chitosan nanospheres loaded plates and evaluate its characteristics such as surface structure and the drug release behavior of the drug in vitro and its anti-Staph aureus efficacy in vitro. Methods: The solvent casting technology was adopted to prepare the gentamicin sulfate/chitosan nanospheres loaded plates. The surface appearance of the plates was observed by SEM,the drug release behavior in vitro was studied by immerse the plates into PBS. And the gentamicin sulfate/chitosan nanospheres loaded plates were placed in the centre of the Mueller-Hinton(MH) agar which were inoculated with the Staph aureus at the concentration of 1 × 108 CFU/ml. The bacteria were cultured on MH agar for 48 h and the diameter of the inhibition zone was measured,and this procedure was repeated until there was no inhibition zone. Result: The result of the SEM shows the gentamicin sulfate/chitosan nanospheres distributed on the surface of plates uniform,the drug release behavior in vitro could continue about 14 days,and the max cumulative release of the drug was about 52%. The anti-Staph aureus efficacy in vitro could continue about 9 days and the max diameter of the inhibition zone was (3.82 ± 0.03) cm. Conclusion: The gentamicin sulfate/chitosan nanospheres loaded plates show well surface appearance and the plates have a steady drug release behavior and significant anti-Staph aureus efficacy in vitro.