The development of smart agriculture has become a critical issue for the future of smart cities, with large-scale management of aquaculture posing numerous challenges. Particularly in the fish farming industry, producing single-sex fingerlings (especially male fingerlings) is crucial for enhancing rearing efficiency and could even provide key support in addressing future global food demands. However, traditional methods of manually selecting the gender of broodfish rely heavily on experienced technicians, are labor-intensive and time-consuming, and present significant bottlenecks in improving production efficiency, thus limiting the capacity and sustainable development potential of fish farms. In response to this situation, this study has developed an intelligent identification system based on the You Only Look Once (YOLO) artificial intelligence (AI) model, specifically designed for analyzing secondary sexual characteristics and gender screening in farmed fish. Through this system, farmers can quickly photograph the fish’s cloaca using a mobile phone, and AI technology is then used to perform real-time gender identification. The study involved two phases of training with different sample sets: in the first phase, the AI model was trained on a single batch of images with varying parameter conditions. In the second phase, additional sample data were introduced to improve generalization. The results of the study show that the system achieved an identification accuracy of over 95% even in complex farming environments, significantly reducing the labor costs and physical strain associated with traditional screening operations and greatly improving the production efficiency of breeding facilities. This research not only has the potential to overcome existing technological bottlenecks but also may become an essential tool for smart aquaculture. As the system continues to be refined, it is expected to be applicable across the entire life cycle management of fish, including gender screening during the growth phase, thereby enabling a more efficient production and management model. This not only provides an opportunity for technological upgrades in the aquaculture industry but also promotes the sustainable development of aquaculture. The smart aquaculture solution proposed in this study demonstrates the immense potential of applying AI technology to the aquaculture industry and offers strong support for global food security and the construction of smart cities.