α-MoO3 is a typical layered semiconductor transition metal oxide, and its unique electronic structure and lattice structure have made it widely studied in recent years. Compared with bulk α-MoO3, α-MoO3 film has excellent optical, electrical and mechanical properties due to its two-dimensional geometric limitations. However, the epitaxial growth of single-layer defect-free α-MoO3 film has not been realized so far. In this paper, molecular beam epitaxy was used in ultra-high vacuum, and a single-layer defect-free semiconductor α-MoO3 film was prepared by van der Waals epitaxial on highly oriented pyrolytic graphite (HOPG) for the first time, and linear defects were generated by hydrogen reduction. The microstructure and apparent energy gap of the defect-free single-layer film and the defect were studied by scanning tunneling microscope (STM). The results show that, high-quality single-layer α-MoO3 films can be prepared on HOPG substrates by accurately controlling the substrate temperature. The film thickness and cell size conform to the characteristics of single-layer α-MoO3, and the apparent energy gap of 1.7 eV is determined by the scanning tunnel spectrum. The high quality of the grown film can be further confirmed by the moire pattern on the substrate HOPG. By introducing hydrogen, bright line defects perpendicular to the moire pattern can be obtained on the surface of the film, and the local apparent energy gap of the line defects is 0.4 eV, that is, a narrowband semiconductor channel is realized inside a broadband semiconductor.