Semiconducting polymer dots (Pdots) have received much attention due to their unique characteristics, including high water solubility, good light stability, excellent biocompatibility, and low cost. Herein, we report a ratiometric nanoprobe based on Pdots-Eu for temperature sensing in vitro. The Pdots-Eu thermometer was composed of a blue temperature-insensitive semiconducting polymer, poly(9-vinylcarbazole) (PVK), a red temperature-sensitive complex tris(dibenzoylmethane)mono(5-amino-1,10-phenanthroline)europium (III) (Eu complex), and an amphiphilic polymer polystyrene graft ethylene oxide functionalized with carboxyl groups (PS-PEG-COOH). The Pdots-Eu thermometer showed two peaks at 368 nm from PVK and 611 nm from the Eu complex. The red/blue fluorescence intensity ratio of Pdots-Eu decreased with an increase in temperature, which could be used for the ratiometric monitoring of temperature change. The results showed that the red/blue fluorescence intensity ratio demonstrated a good linear relationship to the change of temperature from 25 °C to 55 °C. Impressively, the ratiometric probe featured good accuracy and high sensitivity for temperature detection in vitro, which could be used for monitoring temperature change in cells.