The presently claimed invention provides a focusing apparatus and method for a fluorescence microscope which is capable of shortening the time in focusing, increasing system throughput and avoiding from undesirable photo-bleaching. The fluorescence microscope of the present invention employs a portion of excitation light to form images of a sample to determine a focus plane for fluorescence imaging. As intensity of the portion of the excitation light is much higher than that of fluorescence light, the exposure time is highly reduced for image formation used for focusing purpose. The fluorescence microscope of the present invention enables to perform both predictive focusing and multiplex focusing.