A miniaturized fluorescence detection system based on total internal reflection (TIR) configuration, which is applicable to detecting the presence of biological materials labeled with fluorescence dye in micro total analysis systems (${mu}TAS$), is proposed. In conventional fluorescence testing and analysis devices, interference between the excitation light beam and the emitted light from dyes is unavoidable. This paper presents a fluorescence detection system based on TIR configuration that allows the excitation light beam and the emitted light to be spatially perpendicular to each other so as to minimize the interference where fluorescence emission is detected at the orthogonal angle to the excitation beam. We achieved the limit of detection of about 5 nmol/L with a high linearity of 0.994 over a wide range of 6-FAM mol concentration, being comparable to that in earlier studies.