An accurate method for measuring intracellular temperature is potentially valuable because the temperature inside a cell can correlate with diverse biological reactions and functions. In a previous study, we reported the use of a fluorescent polymeric thermometer to reveal intracellular temperature distributions, but this polymer required microinjection for intracellular use, such that it was not user-friendly; furthermore, it could not be used in small cells or cells with a cell wall, such as yeast. In the present study, we developed several novel cationic fluorescent copolymers, including NN-AP2.5 and NN/NI-AP2.5, which exhibited spontaneous and rapid entry (≤20 min) into yeast cells and subsequent stable retention in the cytoplasm. The fluorescence lifetime of NN-AP2.5 in yeast cells was temperature-dependent (6.2 ns at 15 °C and 8.6 ns at 35 °C), and the evaluated temperature resolution was 0.09-0.78 °C within this temperature range. In addition, NN-AP2.5 and NN/NI-AP2.5 readily entered and functioned within mammalian cells. Taken together, these data show that our novel cationic fluorescent polymeric thermometers enable accurate and practical intracellular thermometry in a wide range of cells without the need for a microinjection procedure.