Chitosan (CS) microspheres loaded with levofloxacin (LOF) were prepared by emulsion crosslinking technique and the experimental parameters were optimized by orthogonal tests. The influences of various factors on the combination properties of chitosan microspheres were discussed. The optimal preparation condition was 98000 chitosan molecular weight, 30mg/ml chitosan concentration, 5:1 volume ratio of oil to water, 1:1 molar ratio of amino group of chitosan to aldehyde group of glutaraldehyde (GA), 1500 rpm stirring rate, 2:1 mass ratio of chitosan to levofloxacin and 1 h crosslinking time. The CS microspheres prepared under optimal parameters had spherical morphology with 1.59 μm of average geometric diameter and 0.99 of span, its loading capacity (LC) and association efficiency (AE) values were 21.71% and 80.49% respectively. In vitro release profile showed the levofloxacin released fast initially, and then slowly from the microspheres with T90 occurring at 188.5h and its delivery behavior conformed to First-Order release kinetics model.