〔Abstract〕 Objective To construct a stable monoclonal human embryonic kidney 293(HEK293) cell line ex‑pressing recombinant human coagulation factor Ⅶ （rhFⅦ） and evaluate the expression level and procoagulant bioac‑tivity of rhFⅦ.Methods The plasmid pCDNA3. 1-EGFP-FⅦ was transfected into HEK293 cells to verify the effec‑tiveness of the transfection system. The plasmid pCDNA3. 1-FⅦ was transfected into HEK293 cells, and monoclo‑nal stable transfected cell lines were selected using geneticin(G418). The transcription of the FⅦ gene was identi‑fied by reverse transcription polymerase chain reaction(RT-PCR). The expression level of rhFⅦ in the superna‑tant of the monoclonal stable transfected cell line was detected by sodium dodecyl sulfate-polyacrylamide gel elec‑trophoresis and Western blot. The concentration of rhFⅦ was determined by enzyme-linked immunosorbent assay(ELISA), and the procoagulant activity of rhFⅦ was measured by human coagulation factor Ⅶ potency assay.Results HEK293 cells transfected with pcDNA3. 1-EGFP-FⅦ showed green fluorescence, indicating that rhFⅦ was successfully expressed in the supernatant of HEK293 cells after transient transfection with pcDNA3. 1-FⅦ. The monoclonal stable transfected cell line was obtained by G418 screening. RT-PCR identified that the FⅦ gene was integrated into the genome of the monoclonal stable transfected cell line. The cell viability was good as detected by Cell Counting Kit-8, and a single band of rhFⅦ was obtained by purification of the cell supernatant. The highest rhFⅦ expression was(1. 27±0. 09) mg/L, and the highest procoagulant activity was(380. 29±13. 80)%.Conclusion The monoclonal HEK293 cell lines which can express rhFⅦ protein efficiently and stably with excellent pro‑coagulant bioactivity is successfully screened.