The high production rate generation of drug-loaded biodegradable nanoparticles is a crucial factor in achieving significant and rapid advances in nanomedicine. In this paper, we report a one-step, high yield, coaxial electrohydrodynamic spraying method for generating insulin-loaded polymeric nanoparticles. Poly(lactic-co-glycolic acid) copolymer (PLGA) was used as the model biodegradable polymer. The particle shape and size-distribution were studied using electron and atomic force microscopy and were also subjected to degradation and in vitro insulin release studies for up to five weeks. Nanoparticles as small as 50 nm were produced at the ambient temperature with an encapsulation efficiency of 80 +/- 4.1%, through manipulation of simple process control variables (flow rate, applied voltage and collection point). On spraying, insulin-loaded PLGA nanoparticles were generated without disruption to the insulin structure. By using small amounts of a biocompatible hydrophilic additive, the insulin burst release was reduced by 20% and 15% on the first and second days, respectively, and this was followed by comparatively sustained release over 3 weeks. Coaxial electrohydrodynamic spraying was shown to produce insulin-loaded PLGA, with and without the addition of excipients in a single step; with 85% of particles <100 nm at a polymer concentration of 2 wt%. These insulin-loaded PLGA nanoparticles have the potential to maintain the level of drug release over 3 weeks.