Abstract: To meet the demand for precise detection of adenosine triphosphate (ATP) in biological samples, a "dual-response-reverse" ratiometric fluorescence probe based on cerium-doped carbon dots (CeCDs) and copper nanoclusters (CuNCs) was designed by taking advantage of the fluorescence detection technology, aiming to provide a new solution for the high-sensitivity and anti-interference optical detection of ATP. The synthesis conditions and optical properties of CeCDs and CuNCs were optimized by fluorescence spectroscopy, ultraviolet absorption spectroscopy, transmission electron microscopy and X-ray photoelectron spectroscopy. The construction mechanism of the ratiometric fluorescence probe CeCDs@CuNCs and its optical recognition mechanism for ATP were discussed. Afterwards, CeCDs@CuNCs was applied to the determination of ATP in actual biological samples such as urine and serum. The results showed that the detection range of this method for ATP was 0.5-400 μmol/L, the detection limit was 0.11 μmol/L, and the recovery rate of ATP in urine and serum samples was in the range of 99.3%-106.9%. This method is suitable for the detection of ATP in biological samples.