Stereochemistry plays an important role in the interactions between functional bio-macromolecules and exogenous small-molecule drugs. Ginsenoside Rh2 is an effective constituent of ginseng with chirality of hydroxyl group at carbon-20, which has been found closely related to its multiple biological effects such as anti-tumor activity. However, the stereoselectivity of ginsenoside Rh2 interacting with serum albumin (SA), a vital drug carrier in the body, has been rarely reported. In the present study, interactions between SA and the C-20 eipmers of ginsenoside Rh2 were investigated under simulative physiological condition by molecular docking method and spectroscopic analyses, including UV absorption, fluorescence and synchronous fluorescence spectroscopy. Results showed that both 20S- and 20R-Rh2 could form 1∶1 type non-covalent complex with SA mainly via hydrogen bond and hydrophobic forces. According to the influence on spectra of SA, both epimers led to some increase in hydrophobicity for the light emitting residues of SA, as well as the change in micro-environment around some residues (including tyrosine and tryptophan) responsible for intrinsic fluorescence of SA, and fluorescence quenching mainly by a static mode. However, 20S-Rh2 displayed relatively larger binding constant and free energy in contrast with 20R-Rh2, suggesting stereoselective characteristics of the eipmers. The stereo-selectivity of ginsenoside Rh2 interacted with serum albumin may be related with stereochemistry of C-20.