Intracellular temperature has a fundamental effect on cellular events. Herein, a novel fluorescent polymer ratiometric nanothermometer has been developed based on transferrin protein-stabilized gold nanoclusters as the targeting and fluorescent ratiometric unit and the thermosensitve polymer as the temperature sensing unit. The resultant nanothermometer could feature a high and spontaneous uptake into the HeLa cells and the ratiometric temperature sensing over the physiological temperature range. Moreover, the precise temperature sensing for intracellular heat generation in HeLa cells following calcium ions stress has been achieved. This practical intracellular thermometry could eliminate the interference of the intracellular surrounding environment in cancer cells without a microinjection procedure, which is user-friendly. The prepared new nanothermometer can provide tools for unveiling the intrinsic relationship between the intracellular temperature and ion channel function.