Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) is a highly destructive agricultural quarantine pest, and inhibiting its ovarian development and reproductive capacity represents a key strategy for controlling population expansion.This study focused on key genes regulating ovarian development in female Z. cucurbitae. Through RT-qPCR validation across different tissues—including fat body, midgut, epidermis, Malpighian tubules, ovaries of females, and both sexes (female/male adults)—three key female-reproduction-related genes were identified: the ovary highly expressed gene chorion protein s36 transcript variant X3 (s36), and the ovary-specific genes pickpocket protein 28 (pp28) and fascetto (feo). Their expression patterns throughout the developmental stages of Z. cucurbitae were analyzed. Subsequently, RNA interference (RNAi) was employed to investigate the regulatory roles of s36, pp28, and feo in female reproduction. The results showed that individually silencing s36, pp28, or feo in 3-day-old female adults significantly increased mortality, suppressed ovarian development, and markedly reduced egg production and hatchability. Additionally, the expression of vitellogenin (Vg) was significantly downregulated. This study demonstrates that ovary-specific genes (pp28, feo) and the highly expressed gene (s36) play crucial regulatory roles in the reproduction and ovarian development of female Z. cucurbitae, providing potential molecular targets for eco-friendly pest control strategies.