This study systematically demonstrated the antigenicity kinetics of HBV vaccine microneedles (MNs) during the fabrication, application and storage. To improve the stability of HBsAg in a microneedle patch, several selected saccharides were added to the MN formulations as stabilizers. According to the experimental data, no significant decrease of the bio-activity of HBsAg antigen was found during the microneedle fabrication process. And then immune effects of HBsAg added with different sugars were tested. Chitosan and trehalose loaded HBsAg MNs enhanced the antibody levels to approximately 1.5-fold and 2-fold of the plain HBsAg MNs respectively while sucrose and glucose were not obviously beneficial. During the short-term storage under 60 degrees C, the antigenicity of HBsAg MNs encapsulated with glucose and chitosan declined sharply in 24 h and hardly left after 7 days. As for the groups of HBsAg MNs added with sucrose and trehalose, approximately 90% of HBsAg initial antigenicity maintained, which could be attributed to the protective function of non-reductive disaccharides. As for the long-term storage experiments, the pharmacological activity of HBsAg antigen protected by sucrose and trehalose slightly reduced in 3 months except for the samples under 60 degrees C. In extreme condition, trehalose performed even better protection function than sucrose, of which the antigenicity of HBsAg in MNs left approximately 81% and 63% of its initial, respectively. These results confirmed that trehalose loaded HBsAg MNs enabled stable encapsulation and storage of HBsAg antigen and realized reasonable enhancement of immune effect in a relatively painless, safe, and convenient manner.