Microfluidic cell sorter allows efficient separation of small number of cells, which is beneficial in handling cells, especially primary cells that cannot be expanded to large populations. Here, we demonstrate a microfluidic fluorescence-activated cell sorter (muFACS) with a novel sorting mechanism, in which automatic on-chip sorting is realized by turning on/off the hydrodynamic gating valve when a fluorescent target is detected. Formation of the hydrodynamic gating valve was investigated by both numerical simulation and flow visualization experiment. Separation of fluorescent polystyrene beads was then conducted to evaluate this sorting mechanism and to optimize the separation conditions. Isolation of fluorescent HeLa-DsRed cells was further demonstrated with high purity and recovery rate. Viability of the sorted cells was also examined, suggesting a survival rate of more than 90%. We expect this sorting approach to find widespread applications in bioanalysis.