We propose a method for simultaneous 3D temperature and velocity measurement of a micro-flow field. The 3D temperature field is characterized with two-color laser-induced fluorescence particles which are tracked with micro-digital holographic particle tracking velocimetry. A diffraction-based model is applied to analyze defocused particles to determine the intensity ratio of two fluorescent dyes on the particle. The model is validated with experimental images. As the result shows that the intensity ratio nearly remains unchanged with respect to depth positions, defocused particles can be used as 3D temperature sensors. Numerical work is carried out to check the method, and 3D temperature and velocity field in a 120 μm×120 μm×80 μm test volume are retrieved.