Abstract: In this study, LHCGR(-Ex10)-CRE-luc-HEK293 cell line and LHCGR-CRE-luc-HEK293 cell line were constructed according to the cAMP signaling pathway activated by luteinizing hormone (LH) binding to the receptor. The biological activity of LH was detected by luciferase detection system, and the relative titer of samples was calculated by four parameter fitting analysis. The method conditions were optimized, and the specificity, relative accuracy, precision and linearity of the method were verified. The results showed that there was a dose-response relationship between the concentration of LH and the expression of reporter gene, and it conformed to the four-parameter curve. After optimization, the conditions were determined as follows: the initial concentration of LH was 200 nmol·mL^-1, 6-fold serial dilution, 10 concentrations, the number of cells per well was 30 000, and the incubation time was 6 h. It is proved that the method has strong specificity, good accuracy, precision and linearity. This study successfully established and validated a reporter gene method to detect LH biological activity, which can be used for LH biological activity detection and quality control. With the increasing demand for the research and development of LH biosimilar drugs, the establishment of a stable and simple activity assay method to evaluate the biological activity of LH can provide technical support for the quality control of LH products and a powerful tool for the comparative study of similar products.