Display omitted •Supported lipid bilayers (SLB) from the nucleolipid POP-Ade were prepared.•The SLBs were investigated with QCM-D and neutron reflectometry.•A specific affinity of POP-Ade SLB toward nucleic acids was found. POP-Ade (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyladenosine) is a biocompatible anionic nucleolipid with the DNA nucleoside, Adenosine, in the polar headgroup. We have studied the affinity of nucleic acids of different contour length, composition and structure toward supported lipid bilayers (SLB) composed of POP-Ade mixed with the zwitterionic phospholipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) using quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). In order to highlight the specificity of the nucleic acid interaction, the results were compared with data obtained for SLB containing the anionic phospholipid POPG (1-palmitoyl-2-oleoyl-sn-phosphatidyl-glycerol) replacing POP-Ade. Our results demonstrate that the presence of a nucleobase headgroup provides the bilayers with the ability to bind single stranded nucleic acids in a selective fashion, according to a Watson–Crick pattern. In addition the interaction with double stranded nucleic acids was strengthened. Overall, these findings represent fundamental information for the design of biocompatible DNA vectors with DNA–RNA-based amphiphiles.