We present a novel “Lab-on-DVD” system and demonstrate its capability for rapid and low-cost HIV diagnostics by counting CD4+ cells isolated from whole blood. We show that a commercial DVD drive can, with certain modifications, be turned into an improved DVD-based laser scanning microscope (DVD-LSM). The system consists of a multi-layered disposable polymer disc and a modified commercial DVD reader with rotational control for sample handling, temperature control for optimized bioassay, a photodiode array for detection, and software for signal processing and user interface – all the necessary components required for a truly integrated lab-on-a-chip system, with the capability to deliver high-resolution images down to 1 μm in size. Using discs modified with antibodies, we specifically captured CD4+ cells from whole blood, demonstrating single cell resolution imaging. The novel integrated DVD platform with sub-micron image resolution brings, for the first time, affordable cellular diagnostic testing to the point-of-care and should be readily applicable at resource-limited settings.