Abstract: There is a critical shortage of female broodstock in the yellow catfish industry, making the production of all-female populations using sex-control technology extremely important to meet the demand for seedling production and genetic improvement. In this study, XX genotypic yellow catfish fry were treated with 17α-methyldihydrotestosterone (MDHT) and Letrozole (LZ) through feed during the critical period of sex differentiation (12 to 60 days post-hatching, DPH). Five experimental groups were established: MH (50 mg/kg MDHT), ML (10 mg/kg MDHT), MH+L (50 mg/kg MDHT, 1000 mg/kg LZ), ML+L (10 mg/kg MDHT, 1000 mg/kg LZ), and control group (CR, 0 MDHT, 0 LZ). The ML and ML+L groups obtained the highest proportions of masculinization, at 66.7% and 83.3%, respectively. Sex-reversed individuals in these groups included complete-testis-type (29.2%, 33.3%) and intersex gonads (37.5%, 50.0%), both of which produced 43.8% functional testes. Using XX pseudo-males as fathers and selecting XX females as mothers for multiple generations, 2800000 XX genotypic fry were obtained through induced spawning. In an outdoor earthen pond culture environment, it was found that the ovaries of 98.0% of individuals developed well, while 2.0% of individuals reversed to phenotypic males at 92 DPH. At this point, our research team has established a large-scale technological system for producing all-female yellow catfish populations, providing crucial support for large-scale induced spawning and genetic improvement, and serving as an important guarantee for the healthy development of the yellow catfish industry.