The need is critical and urgent for a real-time, highly specific, and sensitive acute kidney injury biomarker. This study sought to establish a sensitive and specific Miox-NanoLuc transgenic mouse for early detection of drug-induced nephrotoxicity. We generated Miox-NanoLuc transgenic mice with kidney-specific NanoLuc overexpression. Our data showed that Miox-NanoLuc-produced luminescence was kidney-specific and had good stability at room temperature, 4 °C, - 20 °C, and repeated freeze-thaw cycles. Serum levels of BUN and creatinine were significantly increased at day 2 or 3 in cisplatin-treated mice and at day 5 in aristolochic acid (AAI)-treated mice. Particularly, the serum and urine Miox-NanoLuc luminescence levels were significantly increased at day 1 in cisplatin-treated mice and at day 3 in AAI-treated mice. Renal pathological analysis showed that the kidney sections of cisplatin-treated mice at day 5 and AAI-treated mice at day 13 showed cytolysis and marked vacuolization of tubular cells. In conclusion, we developed a new platform to early quantify drug-induced nephrotoxicity before serum BUN and creatinine levels increased and pathological tubular cell injury occurred. This model may serve as an early detection for drug- and food-induced nephrotoxicity and as an animal model to investigate tubular cell injury.