Vanillin is an aromatic compound, which is widely used in food flavoring, beverages, perfumes, and pharmaceuticals. Amycolatopsis sp. is considered a good strain for the production of vanillin from ferulic acid by fermentation; however, its high genomic guanine-cytosine (GC) content (>70%) and low transformation and recombination efficiency limit its genetic modification potential to improve vanillin production. Efficient genome editing of Amycolatopsis sp. has been challenging, but this study developed a CRISPR-Cas12a system for efficient, markerless, and scarless genome editing of Amycolatopsis sp. CCTCC NO: M2011265. A mutant, Δ vdh Δ phdB , was obtained by the deletion of two genes coding byproduct enzymes from the vanillin biosynthetic pathway. The gene deletion increased vanillin production from 10.60 g/L (wild-type) to 20.44 g/L and reduced byproduct vanillic acid from 2.45 to 0.15 g/L in a 3 L fed-batch fermentation, markedly enhancing vanillin production and reducing byproduct formation; the mutant has great potential for industrial application.