This study suggests a new fluorescence microscope to observe micro-samples within fluorophore in a variety of biomedical fields including the fluorescence analysis of a biochip, such as a DNA micro-array. A fluorescence microscope is a device for irradiating light onto a micro-object, executing an excitation and fluorescence emission process. In this study, it adopts a total internal reflection fluorescence(TIRF) method to excite a whole micro-sample substrate different from an existing way which uses an evanescent wave resulting from a total internal reflection on the micro-sample surface. Suggested TIRF microscope can reduce optical noise and obtain images with higher sensitivity thus obtain precise information about the density, quantity, location, etc. of a flurophore, and can simultaneously process separate images even when plurality of fluorophores having different excitation and fluorescent wavelength ranges is distributed, thus easily obtain information about the fluorophores.