Here, we describe a study of screen characterization of aptamers targeting the chiral drug ephedrine using the non-immobilized graphene oxide (GO) SELEX. The improved method of long and short chains was here used to prepare the s5DNA library. The Resonance Rayleigh Scattering (RRS) method was first used to monitor the screening process. Through high-throughput sequencing, the genetic sequence data of 90,487 aptamers were obtained. Through the analysis of the parameters of free energy value and secondary structure prediction model of high repeatability sequence, the 10 candidate sequences were identified. Finally, a best-fit aptamer named EP08 was identified by combining the dissociation experiment. The binding affinity and binding mechanism of the aptamer and target were analyzed using an isothermal titration colorimetry (ITC) experiment and circular dichromatic (CD) experiment. The binding affinity (Kd) of the EP08 aptamer to ephedrine is approximately 2.86 +/- 0.24 mu M. This novel DNA aptamer will help in the future development of a new method for the identification and detection of chiral drug ephedrine. (C) 2019 Elsevier Inc. All rights reserved.