Chiral polyelectrolyte multilayers (PEMs) consisting of poly(L-lysine) (PLL), poly(N-(S)alkylated 4-vinylpyridinium iodide), or poly (ethyleneimine malcose) (PEI-m) as polycations and poly(styrenesulfonic acid) sodium salt (PSS) or poly(vinyl sulfate) as polyanions, as well as a nonchiral PEM composed of poly(ethyleneimine) (PEI) and PSS were deposited on silicon substrates and poly(tetrafluoroethylene) membranes using the layer-by-layer method. For these PEMs, enantiospecific interaction toward one enantiomer of either L/D-glutamic acid (L/D-GLU), L/D-tryptophan, or L/D-ascorbic acid (L/D-ASC), respectively, was studied under variation of the concentration, pH, and ionic strength. Both deposition and enantiospecific interaction were analyzed by attenuated total reflection Fourier transform infrared spectroscopy. Our results show a significant enantiospecific preference of D-GLU over L-GLU at PEMs containing PLL and Of D-ASC over L-ASC at PEMs containing PEI-m. No such enantiospecific preference was found for nonchiral PEMs containing PEI. The enantiospecificity OF PEMs of PLL/PSS toward L/D-GLU could be significantly influenced by the ionic strength and pH values, so that increasing attractive electrostatic interactions resulted in higher enantiospecificity.