In this study, we successfully applied a transition-edge sensor (TES) spectrometer as a detector for microbeam X-ray measurements from a synchrotron X-ray light source in the hard X-ray region to determine uranium (U) distribution at the micro-scale and its chemical species in biotite obtained from a U mine. It is difficult to separate the fluorescent X-ray of the U Lα1 line at 13.615 keV from that of the Rb Kα line at 13.395 keV in the X-ray fluorescence spectrum with an energy resolution of approximately 220 eV using a conventional silicon drift detector (SDD). Meanwhile, the fluorescent X-rays of U Lα1 and Rb Kα were fully separated by a TES with 50 eV energy resolution at an energy of around 13 keV. The successful peak separation by the TES led to an accurate mapping analysis of trace U in micro-X-ray fluorescence measurements and a decrease in the signal-to-background ratio in micro-X-ray absorption near edge structure spectroscopy. Thus, it could be a powerful tool for studying the U distribution and speciation in various environmental samples.