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According to the principle of a three-pinhole mask’s defocused imaging, a set of high-speed defocusing micro-PTV devices was employed for further research on the three-dimensional micro particle tracking velocimetry. The similar characteristics among particles’ corresponding triangle models was used in the process of their defocusing images recognition, and the centroid of particle defocusing image spot was calculated by its gray in two-dimensional Gaussian distribution. As many as 29 sample particles were selected in the process of measurement algorithm analysis about particle’s three-dimensional position, and 29 different depth data were utilized for calibrating in the particles’ depth direction with the method of linear fitting, and the drift compensation functions about the in-plane position of particle was acquired by quadratic polynomial fitting with 870 groups of data. A microscope objective lens of 20′0.4 and high speed CMOS camera were adopted to trace the particles of 2mm diameter in the verification measurement of a long straight micro-channel flow field whose Reynolds number were 0.05 and 0.1 respectively. The results showed that the particle velocity distribution calculated was in good agreement with the numerical simulation curve.