The DNA strand displacement reaction has had sustained scientific interest in building complicated nucleic acid-based networks. However, extending the fundamental mechanism to more diverse biomolecules in a complex environment remains challenging. Aptamers bind with targeted biomolecules with high affinity and selectivity, thus offering a promising route to link the powers of nucleic acid with diverse cues. Here, we describe three methods that allow facile and efficient displacement reaction of aptamers from the living cell surface using complement DNA (cDNA), toehold-labeled cDNA (tcDNA), and single-stranded binding protein (SSB). The kinetics of the DNA strand displacement reaction is severely affected by complex physicochemical properties of the natural membrane. Toehold-mediated and SSB-mediated aptamer displacement exhibited significantly enhanced kinetics, and they completely removed the aptamer quickly to avoid a false signal caused by aptamer internalization. Because of its simplicity, aptamer displacement enabled detection of membrane protein post-translation and improved selection efficiency of cell-SELEX.