To improve the activity of probiotics during oral delivery and to investigate the protective effects on the retina. In this study, multilayer microcapsules encapsulating Lactobacillus rhamnosus YBT20 (YBT20) were prepared using sodium alginate, carrageenan and hyaluronic acid. Then it was characterized by determining the encapsulation rate, particle size distribution and zeta potential, and the results showed that the encapsulation rate of YBT20 by microcapsules was 68.51 % ~ 93.73 %, and the particle size distribution was between 100 and 1000 nm. Meanwhile, the storage stability and antioxidant properties of YBT20 were increased by 38.09 % and 37.01 %, respectively. Furthermore, the in vitro digestion simulation showed that the microcapsules could effectively improve the cell viability of YBT20 in saliva, gastric juice and intestinal juice, and the maximum release rate of YBT20 was 82.0 %. Lastly, the protective effect of microcapsule preparation on retinal damage induced by high-fat diet and blue light was evaluated in SD female rats. The results showed that microcapsule preparation can improve the oxidative damage of retinal pigment epithelium (RPE) in rats, and inhibit IL-6, IL-8, IL-1β and TNF-α. This study provides valuable theoretical information for effective delivery of probiotics and retinal protection.