Abstract: In this study, three types of microspheres were prepared by encapsulating Pediococcus acidilactici (PA) using the gelation method with different concentrations of whey protein isolate (WPI), pectin (PE), and sodium alginate (ALG) as wall materials. The optimal ratio of microsphere wall materials was determined by analyzing the morphological characterization, encapsulation efficiency, and survival of PA after simulated digestion. In addition, the storage stability of the microspheres was evaluated by determining the survival rate of PA after storage under different temperatures and time conditions. Results showed that the ALG-WPI-PE-PA microspheres retained a homogeneous spherical appearance after lyophilization, and the morphology was superior to that of ALG-PA and ALG-WPI-PA microspheres. The encapsulation efficiency of the microspheres was significantly increased (P<0.05) when the WPI concentration was 3.0%, in which the encapsulation efficiency of ALG-WPI-PA was 71.93%, whereas the encapsulation efficiency of ALG-WPI-PE-PA reached 94.33%, which was an increase of 22.4%. In addition, the survival rate of encapsulated PA was significantly increased after simulated in vitro digestion (P<0.05). The optimal concentration of WPI in microspheres was determined to be 3.0%, at which the encapsulation efficiency and survival of PA after digestion were optimized. Storage experiments further confirmed that ALG-3.0% WPI-PE-PA microspheres significantly enhanced the survival rate of PA for a prolonged storage period. This study provides a basis for the further development and application of lactic acid bacteria embedding technology and provides new ideas for the diversification of probiotic preparation products.