Atropisomerism confers unique properties and functions to natural products and synthetic compounds. Recently, biocatalytic approaches for the synthesis of axially chiral molecules have received increased attention due to their ability to achieve exquisite stereocontrol using genetically adaptable catalysts. Here we report the development of an atroposelective desaturation process capable of producing non-C-symmetric biaryls via dynamic kinetic resolution using biocatalysis. Molecular dynamics simulations provide critical insights into protein dynamics during substrate binding and suggest key residues for directed evolution, leading to the optimized catalyst, ADes-5, which exhibits a 70-fold improvement in catalytic efficiency compared with the initial hit. Moreover, the X-ray crystal structure of ADes-5 was determined, providing a detailed view of the enzyme architecture. ADes-5 displays versatility across a broad scope of substrates, yielding biaryl products with excellent yields and enantioselectivities. These findings highlight the synthetic potential of this enzymatic platform and enable further exploration of atroposelective synthesis using enzymes.