This paper aims to develop a three-dimensional (3D) measurement approach to investigate the flow structures of viscoelastic fluid in the curved microchannel by using digital holographic microscope (DHM). The measurement system uses off-axis holographic/interferometric optical setup for the moving target, and the real-time three-dimensional-three-components (3D3C) particle tracking velocimetry (PTV) can be achieved based on the analysis of phase information of holograms. To diagnose the irregular flow inside the microchannel, the 3D temporal positions of tracer particles in the volume of 282 μm × 282 μm × 60 μm have been detected and velocity field was calculated based on the PTV algorithm. Moreover, to explain the flow field inside the curved microchannel, for the first time the polarized high-speed camera was utilized to identify the strong elongation in the viscoelastic fluid. The DHM is proven to be successful for the measurements of microfluidic flow, especially for the truly real-time 3D motions.