This paper reports on a method for detecting three-dimensional particle positions and conducting three-dimensional microflow diagnostics in a microvolume via a three-pinhole defocusing concept. A simple setup and an easy detection method are described. The calibration-based defocusing method is suggested in place of formulae introduced through geometric analyses in previous studies. Depth calibration was performed in a microvolume, and X–Y compensation functions were obtained. By using the calibration functions, three-dimensional particle positions can be calculated at a sub-micron depth resolution. The effects of pinhole masks made with different pattern sizes are also described. The developed method was applied to a microflow in a micro backward-facing step. Time-resolved particle trajectories and three-dimensional volumetric velocity fields at a depth of 50 µm were obtained and are presented here.