A novel technique is reported for counting the number and the percentage of CD4+ T lymphocytes in a polydimethylsiloxane (PDMS) microchannel. This system integrates optical fluorescence detection with resistive pulse sensing enhanced by a metal oxide semiconductor field effect transistor (MOSFET). The MOSFET signal indicates the total number of the cells passing through the detection channel, while the concurrent fluorescence signal records only the number of cells tagged with a specific fluorescent dye. The absolute count of the CD4+ T cells and its percentage to the total lymphocytes can be analyzed by combining the two counting results, which shows comparable accuracy to those from the commercial flow cytometer. The fastest observed counting rate for a single-channel microchip is 8.5 cells per second. This technique is highly promising as it could greatly reduce the cost for HIV diagnosis and treatment and make it accessible to resource-poor developing countries.