The polyethyleneimine (PEI)-based antigen delivery system has been proved valuable for therapeutic vaccines. However, the previous bulk mixing method is not ideal to prepare PEI-base antigen- containing nanoparticles. The wide-size distribution and poor reproducibility limit its further application. In this research, we developed a microfluidic method to prepare nanopolyplexes on chips with Tesla structure to improve the fabrication. The structure and bioactivity of protein were not hampered by the shearing force in microfluidics. Comparison between physiochemical parameters suggested that the polyplexes prepared by Tesla chips were more uniform than those prepared by bulk mixing and non-Tesla chips. The reproducibility was improved obviously. The preparation was not influence much by the operating parameters such as flow rates, reagents concentration, and switching of inlets. The results indicated that it was a robust and reliable method. The data that were obtained from BMDC model demonstrated that the nanopolyplexes with optimal weight ratio had higher antigen cross-presentation efficiency than those in free antigen group. In conclusion, Tesla structured microfluidics offers a better method over bulk mixing in the preparation of PEI-based antigen-containing nanopolyplexes. It greatly expands the scope of our study and increases the potential of PEI-based antigen delivery system.