A novel hydrophilic fluorescence temperature probe (PNDP) based on polarity-sensitive triarylboron compound (DPTB) and PNIPAM is designed and synthesized. In order to overcome the shortcomings of the single-intensity-based sensing mechanism and obtain more robust signals, ratiometric readout is achieved by designing an efficient FRET system (PNDP-NR) between DPTB and Nile Red (NR). PNDP-NR possesses some excellent features, including wide temperature range, good linear relationship, high temperature resolution, excellent reversibility, and stability. Within a sensing temperature range of 30–55 °C, the fluorescence color of PNDP-NR experiences significant change from red to green-blue. PNDP-NR is also introduced into NIH/3T3 cells to sense the temperature at the single-cell level. It gave excellent photostability and low cytotoxicity in vivo.