Abstract: Avian β-Defensin-2 (AvBD2) is an antimicrobial peptide that plays a significant role in the innate immunity of chickens. In this research, the expression vector pPIC9K-AvBD2 was established and expressed in Pichia pastoris. AvBD2 cDNA was cloned from total RNA of chicken lung by Transcriptase-Polymerase Chain Reaction (RT-PCR). The AvBD2 mature peptide gene was amplified from and cloned into a pPIC9K plasmid. Then the recombinant expression vector pPIC9K-AvBD2 was transformed into P. pastoris GS115 to express the fusion protein. The result showed that a recombinant secretion expression vector pPIC9K-AvBD2 was successfully constructed and a fusion protein about 3.9 kDa chicken AvBD2 was expressed. Densitometric scanning analysis demonstrated that the expressed protein exaccounted for about 63.7% of the total secreted bacterial protein. The expression products had antimicrobial activity to Escherichia coli and Staphylococcus aureus in the Assay of Agar Diffusion Method.