Abstract: A single-cell bioanalyzer (SCB) was presented to detect different ovarian cancer cells, i.e. to discriminate NCI/ADRRES cells, which are multidrug resistant (MDR), from non-MDR OVCAR-8 cells. This discrimination has been achieved in the single-cell level by measuring drug accumulation in real-time, in which the accumulation is high in nonMDR single-cells without drug efflux, but is low in MDR single-cells with efflux. The SCB was constructed as an inverted microscope for optical imaging and fluorescence measurement of a cell that was retained in a microfluidic chip. The cell retained in the chip offers sufficient fluorescence signals for the SCB to measure the accumulation of daunorubicin (DNR) in a single ovarian cancer cell in the absence of the MDR inhibitor, cyclosporine A (CsA). The same cell allows us to detect the enhanced drug accumulation due to MDR modulation in the presence of CsA. The measurement of drug accumulation in a cell was achieved after it was captured in the chip, with the correction of background interference. The detection of accumulation enhancement due to MDR modulation by CsA was determined in terms of either the accumulation rate or enhanced amount of DNR in the same single-cell. It showed that with the effectiveness of efflux-blocking by CsA, DNR in a single-cell was increased by 3-fold against its same-cell control. For this single-cell bioanalyzer (SCB), it has the ability to discriminate MDR in different ovarian cells due to drug efflux in them by eliminating the interference of background fluorescence and by using the same-cell control.