Aiming at detecting biological substances accurately and speedy, we are developing a dielectrophoresis-Assisted surface plasmon resonance fluorescence (SPRF) illumination biosensor. A sensing chip of the sensor has Al layers, which are used for electrodes and SPR excitation layers. In this study, we used Escherichia coli (E. coli) stained with 4', 6-diamidino-2-phenylindole (DAPI) as a target substance. First, the structure of the sensing chip was optimized to excite SPR on the Al electrodes by 405-nm light. Then, an emulsion with DAPI-stained E. coli was dropped on the sensing chip and illuminated by a 405-nm laser via a prism. When ac voltages were applied to the Al electrodes, the number of bright spots, due to fluorescence from DAPI-stained E. coli, increased on the electrodes. This result indicates that E. coli, which has a lower permittivity than the emulsion, was collected onto the electrodes where the electric field strength is lowest by negative dielectrophoresis. Therefore, we have succeeded in collecting target biological substances on the surface of the sensing chip and detecting them.