Measuring the changes in tumor cell surface temperature can provide insights into cellular metabolism and pathological features, which is significant for targeted chemotherapy and hyperthermic therapy. However, conventional micro–nano scale methods are invasive and can only measure the temperature of cells across a single plane, which excludes specific organelles. In this study, fluorescence quantum dots (QDs) are functionalized with the membrane transport protein transferrin (Tf) as a thermo‐sensor specific for tumor cell membrane. The covalent conjugation is optimized to maintain the relative fluorescence intensity of the Tf‐QDs to >90%. In addition, the Tf‐QDs undergo changes in the fluorescence spectra as a function of temperature, underscoring its thermo‐sensor function. Double helix point spread function imaging optical path is designed to locate the probe at nanoscale, and 3D thermal imaging technology is proposed to measure the local temperature distribution and direction of heat flux on the tumor cell surface. This novel targeted nanoscale 3D thermometry method can be a highly promising tool for measuring the local and global temperature distribution across intracellular organelles.