A pair of bisbenzimidazole derivatives as catalytic ligand was evaluated in DNA-based hybrid asymmetric reaction. Similar structure of both ligands induced entirely different stereoselectivity in Diels-Alder reaction and Friedel-Crafts alkylation. The binding mode of the two Cu2+ complexes to st-DNA was elaborated by circular dichroism, UV-vis, IR, Raman spectroscopy, and singular value decomposition analysis. The coordination mode of ligand to Cu2+ substantially impacted on dominant conformation of complex. The major binding mode depended on the steric chemistry of complex, directly regulating chiral microenvironment in hybrid catalytic system. Although the title ligand did not present considerable enantioselectivity in catalysis, the current research provided a feasible approach to insight into catalytic microenvironment of chiral scaffold. It will help to reveal the mechanism of stereo-induction in DNA-based hybrid asymmetric reaction. [GRAPHICS] .