The work aims to study the effects of various factors on the loading efficiency and encapsulation efficiency of polyphenol/chitosan/alginate nano-microspheres and research the in vitro release rate of nano-microspheres, so as to provide the preliminary basis for the preparation of slow release antibacterial film at later periods. Through the single factor experiment and orthogonal test, the effects of concentration of alginate, chitosan, CaCl2, polyphenol solutions on the loading efficiency and encapsulation efficiency of nano-particles were investigated, and the in vitro release rate was studied. When the concentrations of alginate, chitosan, CaCl2, polyphenol solutions were 15, 10, 15 and 0.8 mg/mL, the loading efficiency of nano-particles prepared under such process condition was 22.71%, the encapsulation efficiency was 61.38% and the particle size was mainly around 500 nm; the results showed that microspheres had a good sustained release effect. The optimal process preparation conditions obtained lays a good foundation for the preparation of slow release antibacterial film at later periods.