Porphyry Cu-Mo deposits are magmatic-hydrothermal deposits in which sulfide and oxide minerals precipitate from aqueous solutions. However, many questions remain about the composition and evolution of the magmatic-hydrothermal fluids responsible for mineralization. In response to this knowledge gap at the Qulong porphyry Cu-Mo deposit, Tibet, we present a comprehensive major and trace element dataset for biotite (including halogens) from Qulong to elucidate magmatic-hydrothermal fluid compositions and fluid evolution. Based on genesis and occurrence, biotite is divided into primary (igneous), re-equilibrated (igneous modified by hydrothermal fluids), and secondary (hydrothermal) types. All studied biotite grains are Mg-rich, and XMg values (0.59–0.90) increased during fluid evolution, perhaps controlled by high oxygen fugacity (fO2) and sulfur fugacity (fS2) in the magmatic-hydrothermal fluids. The IV(F) and IV(Cl) values and halogen fugacity of biotite indicate that Cl-rich fluids were dominant during early magmatic-hydrothermal evolution, while later fluids were enriched in F. This is consistent with early Cu and late Mo enrichment in the Qulong deposit. We propose a fluid evolution model based on in situ major and trace element data and cross-cutting relationships between the intrusions and the veins. Iron, Ti, Co, Ni, Zn, and Cl contents decreased, while Mg, Si, Al, Sn, Ge, and F contents increased during the evolution of the magmatic-hydrothermal fluid. Importantly, the increase in Fe, Ti, Co, Zn, and Cl and decrease in Mg, Ge, and F contents in hydrothermal biotite as the core of the deposit is approached (extending to ~ 2.5 km depth) may prove to be an important indicator of high-grade mineralized zones. Finally, this study shows that systematic spatial variations in hydrothermal biotite chemistry can potentially be used as a prospecting tool for porphyry deposits worldwide.