The patterned coculture of different types of living cells in a microfluidic device is crucial for the analysis of cellular interactions and cell-cell communication. In the present study, cell patterning was achieved by photocrosslinking benzophenone derivatives in a microfluidic channel. Optimization of UV irradiation conditions enabled successful fixation of live cells. In addition, patterning and co-culture of non-adherent K562 cells and adherent RF-6A cells was achieved by successive rounds of patterning. The present approach is expected to be useful for the development of in vitro methods for studying cell signaling.