The　determination　of　the　dominant　factor　for　the　enantioselectivity　and　reactivity　of　lipase-catalyzed　reactions　of　binaphthyl　compounds　is　of　wide　concern.　To　understand　the　stereo-dependent　behavior　of　2,2-binaphthyl　compounds　towards　lipase,　we　established　a　platform　for　effectively　evaluating　the　reactivity　and　enantioselectivity　of　lipase　towards　binaphthyl　compounds　by　investigating　the　binding　interactions　between　substrate　and　lipase.　Penicillium　expansum　lipase　(PEL)　and　three　2-amino-2′-hydroxy-1,1′-binaphthyl　compounds　(NBs)　with　different　linker　lengths　were　selected　as　the　model　lipase　and　substrates,　respectively,　in　this　study.　The　key　interaction　parameters　such　as　binding　affinities,　binding　modes,　and　thermodynamic　parameters　between　NBs　and　PEL　were　determined　for　the　first　time,　and　the　results　revealed　that　the　substrate-lipase　binding　interaction　is　a　prerequisite　for　successful　lipase-catalyzed　reaction.　The　effect　of　binding　discrepancies　of　NBs　with　different　alkyl　chain　lengths　on　lipase　reactivity　and　enantioselectivity　was　also　unveiled.　From　the　analytical　results　of　NB-PEL　binding　interactions,　a　PEL-catalyzed　two-step　resolution　strategy　was　further　established　and　optimized　for　the　gram-scale　synthesis　of　optically　pure　NB.　The　investigation　on　the　molecular　interaction　between　NBs　and　PEL　showed　great　promise　for　rationally　guiding　the　synthesis　of　binaphthyl　derivatives　that　are　suitable　for　efficient　optical　resolution　by　the　commercially　available　lipase.　©　2018　The　Royal　Society　of　Chemistry.