The in situ mechanical properties of the fibers, matrices and interfaces in an Al2O3 fiber-reinforced β-21S Ti alloy have been evaluated by using two nanoscale indentation tests. The Al2O3 fibers were coated with a refractory metal and Y2O3 duplex coating which served as a diffusion barrier during the HIPing used to produce the metal matrix composites. The hardness of the fibers, interfaces and matrix were obtained by performing a series of indentations across the fiber/matrix interface. The hardness decreases from the Al2O3 fiber to the Ti matrix. Additionally, by doing fiber pushout tests, the interfacial shear strength, interfacial frictional stress and mode II interfacial fracture energy were obtained. Scanning electron microscopy and X-ray mapping were used for microstructural and chemical analysis. The mechanical properties of the interfaces were related to their chemical composition. The interfacial fracture was found to occur at the interface between the refractory metal and the Y2O3. The mode II interfacial fracture energy in this system is more than two orders of magnitude lower than the interfacial fracture energy of Ti/Al2O3 without the diffusion barrier.