Abstract: Cooling-gas injection is crucial to the thermal protection of hypersonic vehicles. As a commonly used coolant, air contains N₂ and O₂, which exhibit different chemical reactivities under temperature conditions where N₂ dissociation is not yet significant. Understanding their behavior in high-enthalpy turbulent boundary layers is of practical importance for vehicle thermal management under realistic flight conditions. Using OpenCFD-Comb, an open-source high-order solver for chemically reacting turbulence developed by our group, direct numerical simulations are performed to investigate species evolution and turbulence–chemistry interactions in a Mach 10 high-enthalpy turbulent boundary layer over a flat plate, with O₂ injected through a slit in the fully turbulent region. Pure O₂ is injected at the free-stream temperature with a blowing ratio of 0.001. Results show that the injection creates a localized oxygen-rich zone downstream, leading to a non-monotonic O₂ distribution and significantly enhanced production of atomic oxygen. Mass-fraction fluctuations of O₂ and N₂ are amplified, while those of N and NO are weakened. The injection strengthens chemical reactions involving O₂, O and NO, but has limited effect on the relatively inactive N₂ and N. Compared with species fluctuations, turbulence–chemistry coupling is more sensitive to temperature fluctuations.