Caragobius urolepis exhibits vital ecological plasticity, living in brackish and freshwater habitats, including the Vietnamese Mekong Delta (VMD). Our observation revealed a notable decline in individual fish per catch due to the increased demands for food supply, which highlights the necessity of effective strategies to ensure long-term fish conservation. This study provided detailed information on testicular traits, sperm-releasing season, and length at first maturity of C. urolepis based on a collection of 354 specimens caught monthly through trawl nets at Dong Hai (Bac Lieu) and Dam Doi (Ca Mau). After dissection, testes were weighed for the determination of gonadosomatic index (GSI) and spawning pattern. Four developmental stages were detected. Analysis of the frequency of occurrence of mature testes and GSI revealed that the sperm-releasing season of C. urolepis was during wet months (with peaks in July, September, and November). The fish first matured at 18.0 cm in Bac Lieu and 18.7 cm in Ca Mau. The findings provided valuable insights for exploiting this species as a wild resource and may contribute to research on artificial reproduction. The study suggests avoiding the exploitation of this fish species during the spawning season to ensure future sustainable exploitation.