The aim of this paper is to develop a digital holographic imaging (DHI) system, capable of capturing three-dimensional (3D) images of aquatic species, with the characteristics of in situ, non-interference, dynamic and 3D detection. The DHI system records the in-line holographic image with a scientific grade CCD, while a camera lens is used to magnify the holographic image. This optical field is retrieved by numerical algorithms, which enable the reconstruction of the field at different distances relative to the detector from a single hologram. Conclusion is drawn that digital holographic sensing and imaging is an appropriate and highly effective method for aquatic microorganism study.