HBscFv-IFN gamma, a fusion protein constructed by fusing gamma-interferon (IFN gamma) with an antibody fragment HBscFv for the purpose of targeted delivery of the cytokine IFN gamma, was designed in order to enhance its therapeutic efficacy through increasing its hepatoma localization. HBscFv and IFN gamma were connected into HBscFv-IFN gamma by the linker (Gly(4)Ser)(3), and then the multicopy recombinant plasmids pPICZ alpha A/(HBscFv-IFN gamma)(1,2,4) were constructed and transformed into Komagatella (Pichia) pastoris X33. The engineering strain X4, which had much higher copy number and could secretively express HBscFv-IFN gamma, was screened from transformed X33 by qPCR. Results from SDS-PAGE, Western blotting and ELISA indicated that HBscFv-IFN gamma displayed an excellent immunoreaction against HBsAg. The culture supernatant of X4 was purified by 14F7 affinity chromatography to obtain the fusion protein HBscFv-IFN gamma in a purity of 95-98%. The HBscFv-IFN gamma was able to bind 27.9% HBsAg in the serum of HBV transgenic mice, showing that the antibody of HBscFv-IFN gamma has high binding affinity against HBsAg. The expressing of the recombinant HBscFv-IFN gamma in P. pastoris provides a promising and inexpensive diagnostic reagent for preventing HBV infection.