A fluorescence detection optical system detects fluorescence beams with two or more different wavelengths and maintains a focal position through an automatic focusing function. A multi-channel fluorescence detection apparatus includes the fluorescence detection optical system. The fluorescence detection optical system includes an automatic focusing unit which receives light reflected off a microfluidic device and determines a focal point by using an astigmatic method or a knife edge method, and an actuator which adjusts a position of an objective lens according to control of the automatic focusing unit. In addition, the fluorescence detection optical system may include a plurality of dual band pass filters, dichroic devices, etc., which provide light beams emitted from at least two light sources and transfer fluorescence generated from the microfluidic device to a photodetector.