Abstract: Aiming at the detection requirements of the microfluidic chip channel and defect detection, a set of reflective image-plane digital holographic microscopic system based on the pre-amplified off-axis optical path was designed and constructed. In the digital holographic microscopy measurement, the phase distortion correction method of the low-spatial frequency objects whose lateral size occupies a relatively large field of view was discussed, and the two-step phase subtraction method was proposed to be more suitable for the phase distortion correction of this type of object. The phase distortion correction effects of two-step phase subtraction method, general polynomials surface fitting method and Zernike high-order polynomials surface fitting method were compared and analyzed through the experiment of a micro-step standard sample with a width of 55 μm and a height of 65 nm. The analysis results show that the relative error of the average height of the micro-step after the distortion is corrected by the two-step phase subtraction method is 1.1%, which is smaller than other methods and has a better distortion correction effect. In addition, the microfluidic chip with a channel width of 80 μm was used as the sample to detect the three-dimensional shape of the micro-channel, the fracture defect and defective defect on the surface of the channel. The quantitative results show that the width of the fracture defect is 14.1 μm and the depth is 431.7 nm. The defective defect has a width of 33.6 μm and a depth of 295.1 nm. The experimental results show the image-plane digital holographic microscopic system provides a new way for the rapid and non-destructive measurement of microfluidic chip microchannels and surface defects, which is of great significance for the quality evaluation of microfluidic experimental systems.