Nicotine is an asymmetric molecule with two enantiomers, namely S-nicotine and R-nicotine.  Studies have shown that different configurations of nicotine have different physiological activities in the body.  Therefore, taking human serum albumin HSA) as the model protein, this paper focuses on the different interaction mechanisms and recognition mechanisms of the binding of S/R-nicotine to HSA.  From the comparative analysis of the results of molecular fluorescence spectroscopy, molecular docking and molecular dynamics simulation it can be seen that interaction exist between S/R-nicotine and HSA.  The dominant force driving the interaction processes between S/R-nicotine and HSA was hydrogen bonding and R-nicotine had a slightly stronger effect on HSA than S-nicotine.  Synchronous fluorescence and three-dimensional fluorescence spectroscopy experiments showed that S/R-nicotine could locate in the active cavity of HSA and this interaction would change the microenvironment around Tyr and Trp residues, slightly affect the α helix structure and loose of the peptide skeleton structure of HSA without affecting the basic skeleton structure of HSA.  In has three-dimensional model of S/R-nicotine-HSA constructed by molecular docking, the most stable configuration was formed by the binding of S/R-nicotine to the subdomain IIA (site I) of HSA, which was confirmed by probe experiments.  Theoretical analysis and experimental results were combined to show that S/R-nicotine had high similarity in their binding modes, binding sites, acting forces and the effects on the spatial structure of HSA.  Therefore, it could be speculated that S/R-nicotine might present consistent blood transport behaviour in vivo.  The investigation on the interactions between S/R-nicotine and HSA is important for the clarification of physiological functions of nicotine entering the bloodstream and also provides experimental and theoretical guidance for its safety evaluation.