During the last few years, fluorescence activated cell sorter has played an important role in a variety of biological investigations as well as clinical diagnostics. However, the conventional fluorescence activated cell sorter has several limitations, such as large size, large sample volumes required for operation, and high cost. In this paper, we present a novel microfluidic device that can separate cells based on various fluorescent protein expression levels. Our system consists of three major parts: focusing, detection, and separation. The operating principles are briefly as follows: first fluorescent cells were delivered into the microfluidic chip and focused in the center of channel by sheath flow. Subsequently, the cells were excited by a 532 nm laser at 30 W and concurrently detected by a photomultiplier tube. Based on their fluorescence intensities, the cells were separated into three outlets by a dielectrophoretic force. Using this system, we successfully separated the genetically modified cells at 0.1 L/min (sample flow rate) to sheath flow rate at 1:5, 5 Vpp voltage, and 800 kHz frequency. The separation efficiency was measured as high as 94.7%. In conclusion, we found that our system has the capability of separating genetically modified cells with various fluorescent intensities and help study biology and medicine in a molecular level.