The AlxGa1-xN epitaxial film is grown on (0001)-oriented sapphire with a 20 nm thick aluminium nitride buffer layer by metal organic chemical vapor deposition. The thickness of AlxGa1-xN layer with high crystal quality is about 2 μrm determined by ultraviolet visible light transmittance spectrum analysis. The homogeneity of aluminium in AlxGa1-xN epitaxial film is tested through the energy Eg at the peak intensity in the deep ultraviolet photoluminescence spectrum. The epitaxial wafer with good homogeneity is used to determine aluminium composition by Rutherford backscattering spectroscopy (RBS). Six samples are measured by tow ion beam analysis laboratories, and the experimental data of RBS random spectrum are simulated by the software. The source of measurement uncertainty is analyzed including the sample homogeneity, pileup correction and counting statistics and so on. The research results show that when the alpha particle is used as incident ion, with 2000 keV energy and 165° scattering angle, the measurement uncertainty of RBS for the determination of aluminium composition (x=0.8) is 2.0% and the coverage factor k=2.