Microfluidic devices can offer precise control for a verity of tasks involving biological specimen. In this paper, we propose an integrated system consisting of a microfluidic device along with a digital holographic microscope and present three-dimensional (3D) sensing and segmentation of biological microorganisms. When the individual microorganisms are inputted into the microfluidic channel, the holographic microscope records their holograms. The holograms are computationally reconstructed in 3D using Fresnel transform and the reconstructed phase images are used to search the position of microorganisms. Optical experiments are carried out and experimental results are presented to illustrate the usefulness of the proposed system.