Doxorubicin (Dox) is a chemotherapy medication used to treat cancer. Herein, we report a rapid and efficient method for detecting Dox in vivo based on a NaGdF₄:Yb³⁺,Er³⁺@NaYF₄ core/shell upconversion nanoparticles (UCNPs) probe. We found that the intensity ratio of green to red emission (IGVRE) bands of the core/shell NaGdF₄:Yb³⁺,Er³⁺@NaYF₄ nanoparticles was sensitive to Dox in blood samples, and drops as the concentration of Dox increases. In addition, the proposed UCNPs probe possessed the advantage that no nanoparticles leaked into the living body, thus overcoming the intrinsic defect (difficulty in removing UCNPs from blood vessels) of the fluorescence resonance energy transfer (FRET) approach. This proposed UCNP probe design and results may provide some guidance for the real-time and efficient detection of Dox, and can be helpful in biomedical applications.