〔Abstract〕 To construct a stable monoclonal human embryonic kidney 293（HEK293）cell line ex- pressing recombinant human coagulation factor VII（rhF Ⅶ ) and evaluate the expression level and procoagulant bioactivity of rhFⅦ . Methods The plasmid pCDNA3. 1-EGFP-FⅦ was transfected into HEK293 cells to verify the effectiveness of the transfection system. The plasmid pCDNA3. 1-FⅦ was transfected into HEK293 cells，and monoclonal stable transfected cell lines were selected using geneticin（G418）. The transcription of the FⅦ gene was identified by reverse transcription polymerase chain reaction（RT-PCR）. The expression level of rhFⅦ in the supernatant of the monoclonal stable transfected cell line was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The concentration of rhF Ⅶ was determined by enzyme-linked immunosor- bent assay（ELISA）， and the procoagulant activity of rhF Ⅶ was measured by human coagulation factor Ⅶ po- tency assay. Results HEK293 cells transfected with pcDNA3. 1-EGFP-FⅦ showed green fluorescence，indicat- ing that rhF Ⅶ 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 rhF Ⅶ 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 procoagulant bioactivity is successfully screened.