The delivery of insulin by non-parenteral routes has gained significant attention over the last two decades. In the present study, we prepared hollow quaternized chitosan microspheres by the SPG membrane emulsification technique and glutaraldehyde cross-linking method. The structural properties, as well as the uniform size and autofluorescence, enabled us to develop oral delivery of insulin which conserved the bioactivity of the encapsulated insulin, achieving bioadhesion of microspheres, increasing the loading ability and optimizing the release profile. In vivo evaluation also saw an optimal reduction in blood glucose level and powerful therapeutic effects after treatment with the designed microspheres, which further confirmed the feasibility of using hollow quaternized chitosan microspheres as insulin carriers for oral administration.