We present a setup of confocal fluorescence microscopy system combined with frequency division multiplexing so that the detection ability of the system is enhanced. In the proposed frequency division multiplexed confocal fluorescence microscopy (FDMCFM) system, fluorescence is excited on the sample (rat neural cell) by two beams of light modulated individually in two arms of the system. Fourier transform, filtration and demodulation are then carried out to restore the fluorescence intensity signal. The FDMCFM system is experimentally conducted, through which the target cells are detected successfully. The experimental results show that the FDMCFM system is convenient for multiple-channels and real-time detection of biological cells′ fluorescence signal. Moreover, it maintains the high spatial and temporal resolution of confocal microscopy.