Temperature is a key indicator of infection and disease, however, it is difficult to measure at a cellular level. Nanoparticles are applied to measure the cellular temperature, and enhancement of the stability and reliability of the signal and higher biocompatibility are demanded. We have developed fluorescent polymeric nanoparticles loaded with temperature-sensitive units (as rhodamine B) and internal reference units (as coumarin) for imaging and ratiometric sensing of the cellular temperature in the physiological range. The fluorescence signal of the nanoparticles was stable in the bio-environment and the ratiometric sensing strategy could overcome the concentration effect of nanoparticles. The nanoparticles were endocytosed by cells and partially presented in mitochondria. The fluorescence intensity ratio of rhodamine B and coumarin using nanoparticles showed good linear correlations in buffer solutions, cell suspensions, and imaging of living cells. Using the fluorescent polymeric nanoparticles, the change of temperature of cells during influenza virus infection could be individually monitored.