Microscopy is an important tool in biology and medicine, but it is often limited to optical imaging structures with high numerical aperture(NA) with a short working distance(wd), for example NA = 0.6 and wd <1 mm are usual. The common microscope objective is inadequate for imaging of living cells in culture as an optical imaging structure with both high numerical aperture and long working distance is required. In this study, a novel optical design has been developed to meet the long working distance and high resolution power imaging of living cells in a vessel with a high culture solution thickness, where cells need to be developed in about 48 hours or a week. The developed optical design was characterized by an ultra-long working distance (wd >13.5 mm) and high numerical aperture (NA = 0.7). This optical imaging system is not only good for the subcellular imaging of free-floating cells in culture, but also for the imaging of cells attached at a surface of vessel.