Quinine　(QN)　and　quinidine　(QD),　the　chief　quinoline　alkaloids　of　various　species　of　cinchona　bark,　are　stereoisomers　to　each　other.　In　this　study,　a　series　of　appropriate　and　efficient　methods　have　been　applied　to　compare　the　binding　modes　of　QN　and　QD　with　bovine　serum　albumin　(BSA).　The　isothermal　titration　calorimetry　and　room　temperature　phosphorescence　results　show　that　both　QN　and　QD　can　interact　with　BSA　at　one　binding　site　to　form　drug-protein　complexes,　mainly　through　enthalpic　driving　force　with　the　binding　affinity　order:　QN＞QD.　The　fluorescence　resonance　energy　transfer　and　time-resolved　fluorescence　spectroscopy　exhibits　that　QN　has　a　larger　energy　transfer　and　more　intensified　binding　capacity　for　BSA　than　QD.　Data　of　dynamic　light　scattering　reveal　that　the　aggregate　state　of　BSA　is　changed　during　this　binding　process,　and　the　particle　size　distribution　of　QN-BSA　bioconjugate　is　larger　than　that　of　QD.　Nuclear　magnetic　resonance　analysis　indicates　that　aromatic　protons　make　more　contribution　during　ligand-protein　complexation　than　that　of　aliphatic　protons.　The　circular　dichroism　spectra　exhibit　different　degrees　of　changes　in　BSA　secondary　structures　in　the　presence　of　QN　and　QD,　respectively.　Copyright　©　2014　John　Wiley　&　Sons,　Ltd.