〔Abstract〕 To perform transient transfection of recombinant human Factor X (rhFX) into HEK293 cells, to optimize transfection parameters, to develop a high-yield in vitro expression system for rhFX production, and to assess the biological activity of expressed rhFX. Methods The eukaryotic expression vector pcDNA3.1-EGFP-FX was constructed by inserting the FX gene into pcDNA3.1-EGFP and subsequently transfected into HEK293 cells to validate the transfection system efficiency. The recombinant expression vector pcDNA3.1-FX was generated through ligation of the FX gene fragment with pcDNA3.1, followed by liposome-mediated transfection into HEK293 cells. The expression of rhFX in the cell supernatant was analyzed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Transfection conditions were systematically optimized, and rhFX concentration was quantified by enzyme-linked immunosorbent assay (ELISA). The coagulation bioactivity of rhFX was evaluated through prothrombin time (PT) assay and chromogenic substrate method. Results Recombinant human Factor X (rhFX) was successfully expressed in the supernatant ofHEK293 cells. Optimal expression was achieved at 80% cell confluence with a ratio of plasmid DNA to transfection reagent as 1:2. Peak rhFX production was observed on day 3 post-transfection. Triplicate ELISA measurements demonstrated a maximum rhFX concentration of 5.20 ng/mL in the supernatant. The prothrombin time (PT) of rhFX-containing supernatant was significantly shorter (P < 0.0001) compared to control supernatant from pcDNA3.1 empty vector-transfected cells. The half maximal inhibitory concentration (IC50) of etoxaban was determined to be 1.449 nmol/L using the chromogenic substrate method based on rhFX, which was in the same order of magnitude as the reported 0.78 nmol/L in the literature. Conclusion HEK293 cells successfully express biologically active recombinant human Factor X (rhFX) protein, laying a foundation for advancing the development of rhFX-based therapeutics.