The mechanisms of retention and separation for protected amino acid (PAA) enantiomers on polysaccharide-based chiral stationary phases (CSPs) under supercritical fluid chromatography (SFC) have yet to be investigated. In this study, 18 pairs of PAAs were firstly enantioseparated using SFC with polysaccharide-based stationary phases, and the enantiorecognition mechanism of PAA on the polysaccharide-based CSPs were explored. The key parameters including the stationary phase, modifiers, additives, column temperature, and back pressure were optimized to achieve the baseline separation of PAA enantiomers, which facilitates a more in-depth investigation into the enantiorecognition mechanism. The ADH chromatographic column demonstrates superior separation capability for PAA enantiomers compared to other types of CSPs and subsequent investigations into the enantiorecognition mechanism were primarily centered on ADH. The calculated thermodynamic parameters indicated that for most enantiomers, the process of enantioseparation was primarily driven by enthalpy; however, for Fmoc-Gln(Trt)-OH specifically, entropy played a more significant role in its separation. Molecular docking study was conducted to explore the enantiorecognition mechanism and the results denoted the important role of hydrogen bond and π-π interactions. In addition, hydrogen bonds play a crucial role in the enhanced retention for PAA enantiomers, as well as in the reversal of the elution sequence among different PAAs. This study provides a valuable reference for the investigation and elucidation of the corresponding separation mechanisms associated with polysaccharide-based CSPs.