Effective delivery system for oral insulin administration is a promising way for diabetes therapy. Herein, we prepared alginate microbeads containing chitosan nanoparticles (CNP) for controlled release of insulin. CNP was developed by reaction between tripolyphosphate (TPP) and chitosan. The ratio of TPP to chitosan was optimized aiming with smaller and more unified distributed CNP. TEM and DLS analysis confirmed that CNP has size around 150 nm with low PDI value and strong surface charge. Encapsulate ability for bovine serum albumin, working as model protein, was 11.45%, and the encapsulate efficiency was 23.70%. To modify the release profile of protein suitable for oral insulin delivery, sodium alginate was applied to coat on the surface of CNP by electrostatic interaction. After that, CaCl(2)was added to reinforce the alginate coating layer. FTIR analysis confirmed the interaction of alginate with chitosan and reaction with calcium ion. After reaction with Ca(2+)ion, size measurement revealed that CNP was incorporated into alginate microbeads with mean diameter about 3.197 mu m. Alginate microbeads presented irregular shape with small particles inside as revealed by optical microscope. Meanwhile, the release test demonstrated that protein release was pH-dependent. Acidic pH value retards protein release and neutral pH value promotes protein release. At last, insulin-loaded alginate microbeads were administrated to hyperglycemia model mice and blood glucose profile was monitored afterward. Insulin-loaded microbeads significantly lowered blood glucose level compared with mice treated with alginate microbeads without insulin. It is noted that insulin-loaded alginate microbeads could lower blood glucose level in much prolonged period of 96 h, indicating that insulin was released in controlled manner.