Abstract: The present invention relates, in general, to a fluorescence microscope and method of observing samples using the microscope and, more particularly, to a fluorescence microscope and method of observing samples using the microscope, which can reduce optical noise and obtain images with higher sensitivity, thus obtaining precise information about the density, quantity, location, etc. of a fluorophore, and which can simultaneously process separate images even when a plurality of fluorophores having different excitation and fluorescent wavelength ranges is distributed, thus easily obtaining information about the fluorophores. The fluorescence microscope of the present invention includes an objective lens, and first and third medium units. The first medium unit has a refractive index of n1 to accommodate one or more micro-objects including fluorophores and provide a path of excitation light to excite the fluorophores.