A cubic silicon carbide (3C-SiC) film was fabricated by pulsed laser deposition on an aluminum nitride layer grown on an off-axis Si(110) substrate. This is the first report about the use of off-axis Si(110) substrates for manufacturing SiC/AlN/Si multilayers. A high-quality epitaxial 3C-SiC(111) film without rotation domains or twins, as evidenced by reflection high-energy electron diffraction (RHEED) measurements, was successfully obtained by using a wurtzite AlN(0001)/off-axis Si(110) substrate. On this 3C-SiC film, graphene was formed through annealing in ultrahigh vacuum at 1200 °C and its structure and chemical bonding were investigated via in situ RHEED and X-ray photoelectron spectroscopy; the full width at half maximum of the C 1s core level peak and the root-mean-square surface roughness were 0.60 eV and 0.14 nm, respectively, which were substantially lower than those (0.73 eV and 1.44 nm) previously reported for a graphene sample with similar thickness but formed on a SiC/AlN/onaxis Si(110) substrate.