Abstract: Hepatitis B is the most common liver infection worldwide. The recombinant Virus Like Particles (VLP) of small Hepatitis B surface antigen (s-HBsAg) vaccine provides excellent protection against Hepatitis B Virus (HBV). In this study, we have generated a construct of Pichia pastoris recombinant which comprises multi expression cassettes encoding s-HBsAg which belongs to HBV B3 genotype predominantly found in Indonesia. An expression cassette containing a synthetic codon optimized open reading frame of HBV B3 genotype/adw serotype s-HBsAg was cloned into pAO815 to generate pAOHBs1 in Escherichia coli. The expression cassette from pAOHBs1 was subsequently isolated and ligated with recombinant previously obtained, generated recombinant plasmid containing 2, 3 and 4 direct repeats of HBsAg expression cassette. The resulted recombinant containing 4 expression cassettes was integrated to P. pastoris genome. Positive integrants and stability during cultivation were identified by PCR and qPCR. The expression of s-HBsAg was induced by methanol and analyzed by SDS PAGE and western blotting. Nucleotide sequencing analysis showed that the inserted fragment encodes amino acids with 100% similarity compared to that designed for HBV B3 genotype. The PCR and qPCR analysis showed that stable P. pastoris integrated with 4 s-HBsAg expression cassette was successfully obtained with methanol utilizing slow phenotype. A protein band with apparent molecular weight which similar to s-HBsAg size was detected based on a SDS PAGE and western blot analysis. A stable integrant of P. pastoris containing four expression cassettes of HBV B3 genotype s-HBsAg capable of producing a vaccine candidate against Hepatitis B was generated.