The existing quantum dot temperature measurement techniques can only measure the planar temperature in the cell but fails in 3D temperature investigation. We present a novel method of measuring the 3D temperature field on nano scale, combining fluorescence spectral characteristics of the CdTe quantum dot probe with optical spatial positioning. Based on dual-helix point spread function, a 3D temperature optical measurement system with a resolution of 0.625 °C is established, providing a new perspective of 3D temperature measurement inside the cell. We thus offer an original research tool for further revealing the evolution process of secretions in cell metabolism.