Enzymatic Baeyer‐Villiger oxidation provides a promising green route utilizing molecular oxygen as the oxidant to produce chiral lactones. Wild‐type (WT) CHMOAcineto leads to enantioselectivity up to 99 % ee (S) in the synthesis of substituted ϵ‐caprolactones. To reverse the inherent enantiopreference of CHMOAcineto toward an array of cyclohexanones with various chain length, we herein reshaped the binding pocket with a minimal number of mutations by a rational design strategy combining “size‐probes” with in silico study, which drastically reduces the screening effort. By probing the binding pocket of variants with different‐sized 4‐substituted cyclohexanones substrates, single, double and triple mutants were identified as the best mutants providing highly reversed enantioselectivity for these probing molecules, respectively. The successful demonstration of the strategy combining “size‐probes” with in silico study in the protein engineering of CHMOAcineto may provide a valuable guidance for facile engineering other BVMOs with customized enantiopreference for the same classified substrates with their substituents on the chiral or prochiral central atom.