Abstract: The gene for coding the fusion protein 3DHSA-Nartograstim was cloned into pPICzαA vector along with the open reading frame of the α-factor signal under the control of the AOX promoter. The recombinant secret expression plasmid was transformed into Pichia pastoris host strain GS115 by electroporation. The analysis of SDS-PAGE demonstrated that the relative molecular masses of 3DHSA-Nartograstim were about 42 kD, and the results of Western blotting proved that 3DHSA-Nartograstim was recognized specially by HSA and G-CSF antibodies. The established expression condition of 3DHSA-Nartograstim was BMMY pH 6. 0, 1. 5% methanol for 84 h, and gray scale scanning showed that the purity of 3DHSA-Nartograstim was 79%. Through the combination of affinity chromatography and hydrophobic chromatography, the purity of 3DHSA-Nartograstim was up to 90% and the final yield was 86 mg/L. The bioactivity of 3DHSA-Nartograstim was measured by NFS-60 proliferation assay. Results demonstrated that 3DHSA-Nartograstim could stimulate NFS-60 proliferation in a dose-dependent manner. The study would be helpful for further research of 3DHSA-Nartograstim.