Particle preloading method by first loading proteins onto nano- or microparticles and then integrating these particles into electrospun polyester nanofibers has been widely used to encapsulate therapeutic proteins into polyester nanofibers. However, poor method design has resulted in unsatisfactory protein delivery performance. For example, the harsh conditions involved in preloading procedures damage the bioactivities of proteins, the improper integration leads to an uneven distribution of particles in nanofibers or insecure attachment of particles to nanofibers, producing uncontrolled protein release profiles. This study aimed to improve the protein delivery performance of polyester nanofibers by rationally designing a particle preloading method. Positively charged chitosan nanoparticles (CNPs) were used as carriers to adsorb negatively charged proteins in mild conditions and as primary barriers for protein release. The polar CNPs were then homogeneously dispersed in a polar polyester solution and subjected to electrospinning. Microscope observations indicated that CNPs were homogeneously embedded within polyester nanofibers. In vitro release behaviour and cell studies showed that proteins retained their bioactivity and could release from polyester nanofibers in a sustained manner for more than 4 weeks without any initial burst. Epidermal growth factor encapsulated in polyester nanofibers enhanced diabetic wound healing in vivo, demonstrating an application potential in biomedicine. Other properties of the nanofibers, including composition, wettability, cytotoxicity, and cell adhesion and spreading, were examined in detail as well.