Aptamers have been widely used in the detection, diagnosis, and treatment of cancer. Owing to their special binding affinity toward cancer-related biomarkers, aptamers can be used for targeted drug delivery or bio-sensing/bio-imaging in various scenarios. The interfacial properties of aptamers play important roles in controlling the surface charge, recognition efficiency, and binding affinity of drug-delivering lipid-based carriers. In this research, the interfacial behaviors, such as surface orientation, molecular conformation, and adsorption kinetics of conjugated AS1411 molecules at different cationic lipid bilayer interfaces were investigated by sum frequency generation vibrational spectroscopy (SFG-VS) in situ and in real-time. It is shown that the conjugated AS1411 molecules at the DMTAP bilayer interface show a higher binding affinity but with slower binding kinetics compared to the DMDAP bilayer interface. The analysis results also reveal that the thymine residues of cholesteryl conjugated AS1411 molecules show higher conformational ordering compared to the thymine residues of the alkyl chain conjugated AS1411 molecules. These understandings provide unique molecular insight into the aptamer-lipid membrane interactions, which may help researchers to improve the efficiency and safety of aptamer-related drug delivery systems.