CE application in aptamer selection (CE-SELEX) shows more advantages than other selection methods. In this study, an online reaction based single-step CE (ssCE) mode was employed for fast obtaining protein-ssDNA complex. Using human thrombin (H-Thr) and its aptamer Apt29 as models, we accomplished the procedures of mixing, reaction, separation, detection and complex collection in single step online process, which took about 10 min to obtain the H-Thr/Apt29 complex. Important factors, affecting the aptamer and H-Thr interaction (buffer, ratio of aptamer and H-Thr amount), and complex separation and collection (voltage and temperature) were discussed. Later, the online reaction of H-Thr with an 80 nt ssDNA library was realized under optimized conditions, and the H-ThrissDNA complex was collected and subjected to PCR. By analyzing the PCR product through capillary gel electrophoresis, the resulting approximative 80 nt DNA length validated the ssDNA sequence in complex. To confirm the availability of ssCE mode, two ssDNA libraries with different lengths (56 nt and 82 nt ssDNA) and three proteins (platelet derived growth factor, PDGF-BB; lactoferrin protein, LF; and single-strand DNA binding protein, SSB) were utilized. Their complex peaks were also observed in electropherograms as expected. Additionally, the online incubation of ssDNA and H-Thr was achieved by stopping the separation voltage for some time when ssDNA passed the H-Thr zone. Our results show the ssCE mode has apparent merits of saving time and sample cost for aptamer selection against protein targets. (C) 2018 Elsevier Inc. All rights reserved.