Real-time monitoring of the distribution of energy released during oxidative phosphorylation (OXPHOS) in living cells would advance the understanding of metabolic pathways and cell biology. However, the relationship between intracellular temperature and ATP fluctuation during the OXPHOS process is rarely studied due to the limitation of the sensing approach. Novel fluorescent polymer probes were developed for accurate simultaneous measurements of intracellular temperature and ATP. Utilizing the fluorescence imaging techniques, it was demonstrated for the first time that the temperature in mitochondria increased 2.4 °C and the ATP fluctuation level simultaneously decreased 75% within 2 min during the OXPHOS process. Moreover, the resultant fluorescent polymer probes had good performance and properties for mitochondrial targeting, providing an effective way for investigating mechanisms by which energy is released during the OXPHOS process.