The increasing use and abuse of antibiotics in agriculture and aquaculture necessitates a more thorough risk assessment. We first advocate a precise assessment that subdivides the assessment scope from interspecies to intraspecific levels. Differences in ENR residues and degradation within the intraspecific category were simultaneously explored. This study chose red and GIFT tilapia, both belonging to the intra-specific category of tilapia, for an enrofloxacin (ENR) exposure experiment. Red tilapia had a lower area under the curve (AUC) representing drug accumulation, indicating a notably shorter withdrawal period (7 days) compared to GIFT tilapia (31.4 days) in the edible parts. While four potential transformation pathways were proposed for ENR in tilapia, red tilapia had fewer detected degradation products (6 items) than GIFT tilapia (10 items), indicating a simpler transformation pathway in red tilapia. Predictive assessments using the Toxtree model revealed that of the four extra degradation products in GIFT tilapia, two may possess carcinogenic and mutagenic properties. Overall, differences were observed in ENR residues and degradation within the intraspecific category, with red tilapia presenting lower risks than GIFT tilapia. This work suggests a new strategy to perfect the methodology for antibiotic risk assessment and facilitate systematic antibiotic administration management in the future. Environmental ImplicationCurrently, the large-scale use of antibiotics in aquaculture poses a serious threat to human and environmental health. While previous studies were limited to the species level, this study takes tilapia as an example to study the residual and metabolic differences caused by intra-species differences for the first time. The findings reveal substantial divergences in the residues and degradation of edible portions within intra-species following antibiotic administration, leading to varying dietary risks. Consequently, conducting a precise assessment of intra-species differentiation research and associated risks could perfect the methodology for antibiotic risk assessment and facilitate systematic antibiotic administration management in the future.