In this study, vancomycin loaded alginate–chitosan composite microspheres were developed by emulsion cross-linking method. Thein vitroand vivo characterizations were done to evaluate the feasibility of application. Our experimental results showed that the emulsification cross-linking technique appeared to be a feasible method for the preparation of alginate–chitosan composite microspheres. The microspheres were spherical in shape and the mean particle size and drug loading were 25.3 ± 5.4μm and 18.5 ± 2.3% respectively. A sustained vancomycin release was realized i.e. the amount of cumulative release increased in a time frame of 24 h to reach an amount i.e. ˜68%. The model that fit best for vancomycin released from the microspheres was the Higuchi kinetic model with a correlation coefficientr= 0.9996.In vivoresults showed that the application of microspheres not only reduced the toxicity, but also maintained effective drug concentration. In addition, no severe signs of epithelial necrosis and sloughing of epithelial cells were detected in histological studies.