In-line digital holographic microscopy is used to obtain a particle size distribution of fly ash after wet electrostatic precipitators. A polydimethylsiloxane microfluidic chip with a cross section of 50 × 200 µm2 is fabricated to offer a suitable experimental environment and constant background. Equivalent Charge Coupled Device pixels are calibrated to be 0.1016 µm, leading a 35.4-times magnification. A sample containing standard particles of 2 µm is first analyzed to confirm the validity and stability of the digital holographic microscopy under flowing condition. The average diameter is reconstructed to be 2.03 µm, leading to a relative standard deviation of 1.5%. Then samples acquired after the wet electrostatic precipitator of a coal power plant is analyzed. Large amounts of holographic pictures are taken and reconstructed to obtain a particle size distribution from 0.5 µm to 3.52 µm. Finally the obtained results are compared with that from a laser particle size analyzer. The acceptable deviation indicates that the method is feasible and accurate to measure fly ash after wet electrostatic precipitators as well as other microparticles.