Both enantiomers of cis‐1,2‐disubstituted cyclohexane have been obtained enantioselectively through engineered amidase‐catalyzed desymmetrization of meso carbocyclic 1,2‐dicarboxamides under mild condition. Based on the enzyme‐substrate binding model suggested by molecular docking, the amplification and reversal of enantioselectivity of amidase was quickly achieved through generating and testing only 8 variants. The origin of enantiopreference for amidase was revealed by MD simulations and the reason for the reversal of enantioselectivity towards amidase variant was disclosed. The synthetic application of biocatalytic meso‐desymmetrization was demonstrated by straightforward transformation of the products to enantiomerically pure heterocyclic compounds.