Prolyl 4-hydroxylase (P4H) is essential to maintain the stable triple-helix structure and function of human collagen 1(α) chain (COL3A1). To obtain hydroxylated human COL3A1, the human COL3A1 and the viral P4H A085R were co-expressed in P. pastoris GS115. The sequence of human COL3A1 without N-terminal and C-terminal was selected for expression. Colony PCR analysis and sequencing after transfection showed that the target gene had inserted successfully. Real-time quantitative PCR (RT-qPCR) indicated that human COL3A1 and P4H were expressed at the mRNA levels. SDS-PAGE and Western blotting analysis of supernatant from the recombinant methylotrophic yest culture showed that recombinant human COL3A1 (rhCOL3A1) was secreted into the culture medium with an apparent molecular mass of approximately 130 kDa. It was noted that the rhCOL3A1 expession quantity was higest at 120 h of induction. Furthermore, mass spectrometry analysis demonstrated that the rhCOL3A1 was expressed successfully. His-tagged rhCOL3A1 protein was purified by Ni-affinity column.