The present study addresses the challenges of uncontrolled temperature and pollution in aquatic environments, with a focus on fish ability to tolerate high temperatures. The investigation aimed to determine the role of iron nanoparticles (Fe-NPs) in enhancing the thermal tolerance of Pangasianodon hypophthalmus exposed to high-temperature stress, arsenic (As), and ammonia (NH3) toxicity. Fe-NPs were synthesized using green approaches, specifically from fish gill waste. The dietary Fe-NPs were formulated and administered at 10, 15, and 20 mg kg⁻1 of feed. Notably, Fe-NPs at 15 mg kg⁻1 of diet significantly reduced the critical thermal minimum (CTmin) to 14.44±0.21 °C and the lethal thermal minimum (LTmin) to 13.46±0.15 °C, compared to the control and other treatment groups. Conversely, when Fe-NPs at 15 mg kg⁻1 were administered with or without exposure to stressors (As+NH3+T), the critical thermal maximum (CTmax) increased to 47.59±0.16°C, and the lethal thermal maximum (LTmax) increased to 48.60±0.37 °C, both significantly higher than the control and other groups. A strong correlation was observed between LTmin and CTmin (R2 = 0.90) and between CTmax and LTmax (R2 = 0.98). Furthermore, dietary Fe-NPs at 15 mg kg⁻1 significantly upregulated the expression of several stress-related genes, including HSP70, iNOS, Caspase-3a, CYP450, MT, cat, sod, gpx, TNFα, IL, TLR, and Ig. The enhanced thermal tolerance (LTmin and LTmax) can be attributed to these gene regulations, suggesting the mechanistic involvement of Fe-NPs in improving thermal resilience. Overall, the findings demonstrate that dietary supplementation with Fe-NPs, particularly at 15 mg kg⁻1, improves thermal tolerance and stress response in P. hypophthalmus by enhancing gene expression and overall thermal efficiency under stressor conditions.