In the present work, we aimed to develop alginate-coated chitosan nanoparticles for oral insulin delivery. The N-[(2-hydroxy- 3-trimethylammonium)propyl] chitosan chloride (HTCC) was synthesized, and the quatemized chitosan nanoparticles (HTCC-T NPs) were prepared by ionic gelation of HTCC using tripolyphosphate (TPP). The alginate-coated quatemized chitosan nanoparticles (HTCC-A NPs) were prepared by coating HTCC-T NPs with alginate (ALG) solution under mild agitation. Particle size, zeta potential, surface morphology, drug loading and entrapment efficiency of HTCC-A NPs were characterized using Zeta-sizer, TEM and HPLC assays. It was found that HTCC-A NPs exhibited uniform spherical particles with the size of (322.2±8.5) nm and positive charges (14.1±0.6) mV. Our data showed that the release behavior of HTCC-A NPs was quite different from that of HTCC-T NPs (without ALG coating) when incubated with various medium at different pH values in vitro, suggesting that ALG coating over the HTCC-T NPs improved the release profile of insulin from the NPs for a successful oral delivery. The ALG coating could also improve the stability of insulin against enzymatic degradation. From circular dichroism spectrum, it was revealed that HTCC-A NPs were capable of maintaining the conformation of insulin. The relative pharmacological bioavailability of HTCC-A NPs was 8.0%±2.5% by intraduodenal administration. The HTCC-A NPs significantly increased (P〈0.05) the relative pharmacological availability (2.2 folds) compared with HTCC-T NPs after oral administration. HTCC-A NPs significantly enhanced the in vivo oral absorption of insulin and exhibited promising potentials for oral delivery.