The solvation of DNA in water facilitates the formation of a hydration layer surrounding it， thus stabilizing the DNA duplex in the biological aqueous environment. In this study， via using the lipid bilayer as a soft substrate to accommodate the duplex oligonucleotide， the structure of the water layer surrounding the oligonucleotide was detected under the perturbation of the calcium ions (Ca) with chiral and achiral sum frequency generation (SFG) vibrational spectroscopy. With increasing Ca concentration， both the chiral and achiral water vibrational signals had similar concentration-dependent changes， i.e.， an initial decreasing phase followed by an increasing phase. However， when the Ca concentrations were adjusted to within the range comparable to those in the human serum， the chiral water vibrational signals remained nearly unchanged， whereas the achiral water vibrational signals still changed as a function of the Ca concentration. Therefore， the current experimental result supports the possible protection function of the chiral hydration layer against the Ca ions， which generally exist in the cell sap and play important roles in many biological functions.