Abstract This paper presents a new strategy for sorting fluorescently labeled cells with a microfluidic device. Cell separation was performed with flow switching using changes in fluidic resistance in a waste channel caused by sol–gel transition of a thermo-reversible gelation polymer (TGP). The TGP is liquid at room temperature and turns into a gel state upon heating. The sol–gel transition of the TGP was induced by heating with a 1480 nm laser light for about 3 ms. When a fluorescence signal emitted from a target particle was detected, the waste channel is heated with the laser to increase the flow resistance of the channel. This permits the target particles to be sorted and transferred to the collection channel. In contrast to a previously reported TGP-based sorter, the sample sorted using the microfluidic cell sorter of the present study does not have to be mixed with TGP solution, and the sorted sample is suitable for following experiment. The performance of the cell sorter was evaluated by separating fluorescent microspheres. Two kinds of fluorescent microspheres were separated with a recovery ratio and purity of about 90%. As an application of the cell sorter, Escherichia coli cells expressing the green fluorescent protein (GFP) were separated from those expressing the Discosoma sp. red fluorescent protein (DsRed). About 17,000 cells were sorted with a 75% recovery ratio and 90% purity at a throughput of about 5 cells/s.