To evaluate the biocompatibility of the theophylline/chitosan/β-cyclodextrin microspheres, which has a potential application in pulmonary delivery system. The detection of LDH and protein in BALF was examined acute cell toxicity, hemolysis test was carried out to estimate blood toxicity; Micronucleus Test was reckoned to identify genotoxicity, MTT assay was used to evaluate in vitro cytotoxicity, and muscle implantation investigated the tissue biocompatibility. The results demonstrated that the total contents of protein and LDH in BALF were not significantly different from that of normal group. The experiments showed that the cytotoxicity was depended on the concentration and had no cytoxicity at low concentration and no hemolysis activity. The micronucleus frequency of MS B was 0.99‰, which showed no genotoxic effects either. The results of implantation showed that the microspheres had no effect on hemoglobin and no toxicity in the liver and kidney. The inflammations of muscle tissue were not significantly different from that of operative suture, therefore, the MS B possess high good biocompatibility and can be applied in pulmonary sustained release systems.