To fabricate a controlled release system for icariin for application to tissue engineering, gelatin/hyaluronic acid (Gel/HA) composite microspheres loaded icariin were prepared through emulsion-coagulation method using glutaraldehyde (GA) as cross-linker. The free Gel/HA and Gel/HA-icariin microspheres were characterized by Fourier transforms infrared (FTIR) and scanning electron microscopy (SEM). The influences of cross-linker content on the drug loaded and encapsulation efficiency of the Gel/HA microspheres were investigated, and the effects of cross-linker content, cross-linking time and loaded drug content on the release behavior of icariin in vitro were also studied. Moreover, the in vitro release kinetics of icariin from Gel/HA microspheres was analyzed. The results showed that both Gel/HA and Gel/HA-icariin microspheres are spherical with size ranged from 20 to 60 mu m, and the Gel/HA-icariin microspheres showed rougher surface than Gel/HA microspheres. The loaded efficiency and the encapsulation efficiency decreased with increasing cross-linker content. The cumulative release of icariin from the Gel/HA microspheres decreased with increasing GA content and cross-linking time; however, the cumulative release increased with increasing icariin content in the microspheres. The results of icariin release kinetics from Gel/HA microspheres indicated Fickian diffusion. We concluded that it was feasible for Gel/HA to act as carriers of slow release of microspheres, and the release rate of icariin from the microspheres was adjustable.