The endemic lempuk fish (Gobiopterus sp.), inhabiting Lake Ranu Grati, East Java, Indonesia, is increasingly threatened by environmental degradation and anthropogenic disturbances, highlighting the need for reliable biological indicators to assess ecosystem health and support conservation strategies. This study evaluated physiological stress biomarkers as indicators of environmental stress and investigated their relationship with fish health and reproductive performance to support ecological monitoring, domestication, and F1 seed production of Gobiopterus sp. Fish were collected from two sampling sites representing contrasting environmental conditions in Lake Ranu Grati, where water quality and physiological responses were simultaneously assessed. Physiological stress biomarkers were assessed using Reactive Oxygen Species (ROS), Superoxide Dismutase (SOD), Catalase (CAT) and Cortisol. Reproductive performance was evaluated based on Fertilization Rate (FR), Hatching Rate (HR), and Survival Rate (SR). Relationships among variables were analyzed using Spearman correlation and Principal Component Analysis (PCA). The results demonstrated that fish collected from the environmentally less favorable site exhibited higher ROS, SOD, CAT, and Cortisol levels accompanied by lower fertilization, hatching, and survival rates. Correlation analysis showed that reproductive performance declined with increasing physiological stress, whereas broodstock health parameters remained within physiological tolerance limits. Further PCA analysis differentiated physiological responses across sampling sites, with ROS and cortisol making the largest contributions to the observed variation. These findings demonstrate the potential of physiological biomarkers as early indicators of environmental quality decline and provide scientific support for the ecological monitoring, domestication, and conservation of the endemic Gobiopterus sp.