As a precise and versatile tool for genome manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform holds promise for modifying fish traits of interest. With the aim of reducing transgene introgression and controlling reproduction, upscaled disease resistance and reproductive intervention in catfish species have been studied to lower the potential environmental risks of the introgression of escapees as transgenic animals. Taking advantage of the CRISPR/Cas9-mediated system, we succeeded in integrating the cathelicidin gene (As-Cath) from an alligator (Alligator sinensis) into the target luteinizing hormone (lh) locus of channel catfish (Ictalurus punctatus) using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock in (KI) efficiency (22.38%, 64/286) but low on-target events was achieved using the ssODN strategy, whereas adopting a dsDNA as the donor template led to an efficient on-target KI (10.80%, 23/213). The on-target KI of As-Cath was instrumental in establishing the lh knockout (LH–_As-Cath+) catfish line, which displayed heightened disease resistance and reduced fecundity compared with the wild-type (WT) sibling fish. Furthermore, administration of human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone analogue (LHRHa) can restore the reproduction of the transgenic fish line. Overall, we replaced the lh gene with an alligator cathelicidin transgene and then administered hormone therapy to gain complete reproductive control of disease-resistant transgenic catfish in an environmentally sound manner. This strategy not only effectively improves consumer-valued traits but also guards against unwanted introgression, providing a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.