The influence of thermal annealing on the properties of germanium grown on silicon (Ge-on-Si) has been investigated. Depth dependencies of strain and photoluminescence (PL) were compared for as-grown and annealed Ge-on-Si samples to investigate how intermixing affects the optical properties of Ge-on-Si. The tensile strain on thermally annealed Ge-on-Si increases at the deeper region, while the PL wavelength becomes shorter. This unexpected blue-shift is attributed to Si interdiffusion at the interface, which is confirmed by energy dispersive X-ray spectroscopy and micro-Raman experiments. Not only Gamma- and L-valley emissions but also Delta(2)-valley related emission could be found from the PL spectra, showing a possibility of carrier escape from Gamma valley. Temperature-dependent PL analysis reveals that the thermal activation energy of Gamma-valley emission increases at the proximity of the Ge/Si interface. By comparing the PL peak energies and their activation energies, both SiGe intermixing and shallow defect levels are found to be responsible for the activation energy increase and consequent efficiency reduction at the Ge/Si interface. These results provide an in-depth understanding of the influence of strain and Si intermixing on the direct-bandgap optical transition in thermally annealed Ge-on-Si.