Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed on activated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibits proliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involved in antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain was amplified and cloned into expression plasmid pGEX-5x-3. The fusion protein GST-PD-1 was effectively expressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to obtain bioactive soluble GST-PD-1. Fusion protein of above 95% purity was acquired by a convenient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependent in vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-L1 and PD-1 and increase the production of IL-2 and IFN-gamma of phytohemagglutinin-activated T cells.