Understanding of the molecular relationships in carbohydrate-protein interactions provides useful information on biological processes in living organisms and is also helpful for development of potent biomedical agents. Herein, the interaction unbinding force between GM1 pentasaccharide and Vibrio cholera toxin (ctx) proteins was measured using atomic force microscopy (AFM), which enabled us to determine the interaction of ctx holotoxin (ctxAB) with GM1 and the interactive formation. First, the interaction force measured between A and B subunits (ctxA-ctxB) was 184.2 ± 4.5 pN, and the unbinding forces were evaluated to confirm the role of ctxA in ctxAB complex formation and were determined to be 443.7 ± 7.5 and 535.7 ± 25.9 pN for GM1-ctxB and GM1-ctxAB complexes, respectively. The force difference of ∼90 pN between GM1-ctxB and GM1-ctxAB might be due to the formation of the cholera toxin complex. Importantly, from the analogue analyses, we understand how structural and binding positional differences in complex carbohydrates affect the interaction with protein and surmise that the GM1-ctxAB complex makes a "two-finger grip" formation through the conformational change of a flexible carbohydrate. In conclusion, using AFM force analysis, we successfully quantified and characterized the interactive configuration of carbohydrate-protein molecules.