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Insulin therapy has been the best choice for the clinical management of diabetes mellitus. The current insulin therapy is via subcutaneous injection, which often fails to mimic the glucose homeostasis that occurs in normal individuals. Oral delivery is the most convenient administration route. However, insulin cannot be well absorbed orally because of its rapid enzymatic degradation in the gastrointestinal tract. Therefore, nanoparticulate carriers such as polymeric nanoparticles are employed for the oral delivery of insulin. This study aims at the statistical optimization by Box-Behnken statistical design, fabrication by ionic gelation technique and in vitro characterization of insulin nanoparticles containing thiolated N- dimethyl ethyl chitosan (DMEC-Cys) conjugate. Independent variables such as the concentrations of polymer, TPP and insulin were optimized using a 3-factor, 3-level Box–Behnken statistical design. The selected dependent variables were size, zeta potential, PdI and associated efficiency of nanoparticles. The optimized nanoparticles were shown to have mean particle size diameter of 148 nm, zeta potential of 15.5 mV, PdI of 0.26 and AE of 97.56%. In vitro release study, FTIR, FE-SEM and cytotoxicity also indicated that nanoparticles made of this thiolated polymer are good candidate for oral insulin delivery.