In an alginate/chitosan nanoparticle system, insulin was protected by forming complexes with cationic β-cyclodextrin polymers (CPβCDs), which were synthesized from β-cyclodextrin (β-CD), epichlorohydrin (EP) and choline chloride (CC) through a one-step polycondensation. Due to the electrostatic attraction between insulin and CPβCDs, as well as the assistance of its polymeric chains, CPβCDs could effectively protect insulin under simulated gastrointestinal conditions. The nanoparticles have their mean size lower than 350 nm and can load insulin with the association efficiency (AE) up to 87%. It is notable that the cumulative insulin release in simulated intestinal fluid was significantly higher (40%) than that without CPβCDs (18%) because insulin was mainly retained in the core of the nanoparticles and well protected against degradation in simulated gastric fluid. Far-UV circular dichroism analysis also corroborated the preservation of insulin structure during the nanoparticle preparation and release process.