Transcutaneous immunization (TCI) has many advantages compared with needle-based administrations. But the conventional TCI shows poor permeation of antigens across the skin barrier. In this study, Functional MicroArray (FMA) system was used to poke the skin and increase the permeability, and the hydrogel patch formulation was used as the carrier for transdermal delivery of hepatitis B surface antigen (HBsAg) and cholera toxin B (CTB) as an adjuvant. In vitro permeation of antigen was studied using porcine ear skin and rat abdominal skin. The results showed that FMA system could significantly increase the permeation of HBsAg across skin compared with conventional TCI. HBsAg loaded hydrogel formulation exhibited better antigenic thermostability than the liquid formulation. In vivo immunization studies were performed in mice, and the serum IgG titer, IgG2a/IgG1 ratio were measured. The results showed that TCI with FMA induced more potent immune responses than the groups without FMA pretreatment. CTB adjuvanted TCI group could induce higher IgG titers compared with the group without CTB. Furthermore, TCI group can maintain a longer duration of stable IgG titers compared with the intramuscular injection (IM) group. In conclusion, the FMA/hydrogel system was proved to be a potential vaccination strategy against hepatitis B virus.