The development of non-injection route for protein drugs, especially oral administration, has been the main focus of controlled release of drugs. To overcome obstacles unsolved such as enzyme degradation and penetration barrier of intestinal epithelium, technologies using microspheres as carrier of protein drugs have been proven potential to realize oral administration. It has been demonstrated that microspheres can not only protect proteins, but also facilitate the penetration and absorption through Peyers patches when the size is smaller than 10 μm. Therefore, the objective of this paper is to prepare protein-loaded microspheres with size ≤10 μm. Electrostatic droplet generation technology was used with insulin and hemoglobin as drug models and sodium alginate as microsphere material. By decreasing the surface tension of feed solution by adding surfactant, and improving electric field distribution by changing the shape of container and electrode for gelation solution, protein-loaded microspheres with mean size less than 10 μm were successfully produced through needle with diameter of 400 μm. The microspheres showed good sphericity and narrow size distribution. The mean standard variance of size distribution was 1.61. The encapsulation efficiency of proteins was over 70%. Moreover, the significance analysis of factors influencing the size of protein loaded microspheres was carried out through orthogonal experiments, which showed that output voltage (U), needle diameter (D) and the distance between needle tips to the surface of gelation solution (δ ) influenced significantly the size of microspheres. Finally, the statistic analysis showed that when confidence level wasα=0.05, and α=0.1, confidence interval of microsphere size can be (6.2545, 10.1735) and (6.6022, 9.8258) correspondingly, suggesting that there is good repeatability and reliability for improving electrostatic droplet generation technology to prepare protein-loaded microspheres with size ≤10 μm.