This paper aims to develop a three-dimensional measurement approach to investigate the flow structures of viscoelastic fluid in the curved microchannel by using digital holographic microscope (DHM). With the advantage of DHM, the real-time three-dimensional measurement for the complex flow field can be accomplished. The measurment system uses off-axis holographic / interferometric optical setup for the target, and 3D3C particle tracking velocimetry (PTV) can be achieved based on the analysis of phase information of holograms. To diagnose the chaotic flow inside the microchannel, the 3D temporal positions of tracer particles in the volume of 282μm × 282μm × 60μm have been detected and real-time velocity vectors were calculated based on the PTV algorithm. The measured flow field was then compared with the results obtained by using confocal micro particle image velocimetry (PIV). This technique is proven to be successful for the measurements of microfluidic flow, especially for the truly real-time 3D motions.