Genipin-crosslinked complex microspheres made of the combination of two polymers, ethyl cellulose and chitosan, were prepared by spray drying method. Rifabutin, an anti-tuberculosis agent was used as a model drug. Effects of various specifications of ethyl cellulose and chitosan, different drug/polymers ratios and crosslinking effect of genipin on complex microspheres and drug release characteristics were compared to obtain optimized manufacturing parameters. The complex microspheres showed a significant different shape as compared to chitosan microspheres. Biphasic release features were observed in the in vitro and in vivo release study, consisting of an initial quick release phase and an extended sustained release phase. Furthermore, pulmonary drug concentrations of rats after administering the complex microspheres were maintained on a therapeutic level for at least 24 days.