The microfluidic platform is an important tool for diagnosis and biomedical studies because it enables us to handle precious cells and infectious materials safely. We have developed an on-chip microfluidic sorter with fluorescence spectrum detection and multiway separation. The fluorescence spectrum of specimens (495-685 nm) in the microchannels was obtained every 2 ms using a 1 x 16 arrayed photomultiplier tube. The specimen was identified by its spectrum and collected into the corresponding channel based on our previously reported thermoreversible gelation polymer technique (Y. Shirasaki, J. Tanaka, H. Makazu, K. Tashiro, S. Shoji, S. Tsukita and T. Funatsu, Anal. Chem., 2006, 78, 695-701). Four kinds of fluorescence microspheres and three kinds of Escherichia coli cells, expressing different fluorescent proteins, were successfully separated with accuracy and purity better than 90% at a throughput of about one particle per second.