Abstract: This study employed CHO-K1-FSHR-CRE-Luc transgenic cells as target cells to detect the biological activity of rhFSH-CTP using a luciferase detection system. Key parameters of the method were optimized, and its performance was validated according to the requirements of the 2020 Edition of Pharmacopoeia of the People's Republic of China (Vol IV), including relative accuracy, intermediate precision, linearity and range, and reliability results. The results showed that rhFSH-CTP conformed to the four-parameter equation: y = (A-D)/1+(x/C)^B+D, exhibiting an excellent dose-effect curve. The geometric means of relative biological activities (n = 8) at five titer levels (50%, 80%, 100%, 125%, and 200%) were (53.87 ± 2.47)%, (81.58 ± 1.66)%, (96.76 ± 4.64)%, (127.03 ± 8.19)%, and (207.62 ± 15.52)%, respectively. The relative bias (RB) and its 90% confidence intervals were 7.7% (4.2%, 10.8%), 2.0% (0.6%, 3.3%), -3.2% (-6.4%, -0.3%), 1.6% (-2.9%, 5.8%), and 3.8% (-1.6%, 8.9%), with absolute values of RB and confidence intervals all less than 11%. The upper limits of geometric coefficients of variation (GCV) and their confidence intervals for five titer levels were less than 20%. The linear range spanned 50%-200%, with a slope of 0.974 2 and R² = 0.994 8, meeting the criteria of General Chapter 9401 in Volume IV of the 2020 Pharmacopoeia. Additionally, the deviation parallelism and regression terms satisfied the reliability test requirements of General Chapter 1431 (biological assay statistics) in the same pharmacopoeia edition. In conclusion, this study successfully established a method for detecting the biological activity of rhFSH-CTP, which demonstrates good validity and reliability.