This study demonstrates the first intracellular temperature mapping using a fluorescent polymeric thermometer (FPT) and fluorescence lifetime imaging microscopy (FLIM). The FPT, with a hydrodynamic diameter of 8.9 nm and high hydrophilicity, was designed to diffuse throughout the cell and respond to temperature changes. The fluorescence lifetime of FPT was used as a temperature-dependent variable, allowing for accurate temperature measurement independent of concentration and environmental factors. The spatial and temperature resolutions of the thermometry were at the diffraction limited level (200 nm) and 0.18–0.58 °C, respectively. Intracellular temperature distributions revealed significant temperature differences between the nucleus and cytoplasm, with the nucleus showing a higher temperature than the cytoplasm. These differences were found to be cell cycle-dependent, with the nucleus being warmer in G1 phase cells compared to S/G2 phase cells. Additionally, the centrosome and mitochondria were observed to have localized heat production, with the centrosome being significantly warmer than the cytoplasm and mitochondria showing increased temperature near their locations. The study also confirmed the functionality of FPT in HeLa cells, demonstrating universal characteristics among mammalian cells. This intracellular temperature mapping provides insights into the intrinsic relationship between temperature and organelle function, offering potential for better understanding cellular processes and advancing medical applications.This study demonstrates the first intracellular temperature mapping using a fluorescent polymeric thermometer (FPT) and fluorescence lifetime imaging microscopy (FLIM). The FPT, with a hydrodynamic diameter of 8.9 nm and high hydrophilicity, was designed to diffuse throughout the cell and respond to temperature changes. The fluorescence lifetime of FPT was used as a temperature-dependent variable, allowing for accurate temperature measurement independent of concentration and environmental factors. The spatial and temperature resolutions of the thermometry were at the diffraction limited level (200 nm) and 0.18–0.58 °C, respectively. Intracellular temperature distributions revealed significant temperature differences between the nucleus and cytoplasm, with the nucleus showing a higher temperature than the cytoplasm. These differences were found to be cell cycle-dependent, with the nucleus being warmer in G1 phase cells compared to S/G2 phase cells. Additionally, the centrosome and mitochondria were observed to have localized heat production, with the centrosome being significantly warmer than the cytoplasm and mitochondria showing increased temperature near their locations. The study also confirmed the functionality of FPT in HeLa cells, demonstrating universal characteristics among mammalian cells. This intracellular temperature mapping provides insights into the intrinsic relationship between temperature and organelle function, offering potential for better understanding cellular processes and advancing medical applications.