Abstract: To select the Aptamer (APT) of Zonula Occludens-1 (ZO-1), a systematic evolution technique of ligands was used based on exponential enrichment (SELEX), and 15 APT_ZO-1 were selected as Aptamers finally. Excluding linear and similar structures, an enzyme-linked immune-sorbent assay was used to evaluate the affinity between 9 APTs_ZO-1 and ZO-1. The analysis results show the order of affinity, APT_ZO-1 8 > APT_ZO-1 2 > APT_ZO-1 7 > APT_ZO-1 13 > APT_ZO-1 1 > APT_ZO-1 5 > APT_ZO-1 15 > APT_ZO-1 12 > APT_ZO-1 4, signed with Cy5 fluorescent labeling, for instance, Cy5-APT_ZO-1 8 labeling for APT_ZO-1 8. The enzyme-linked immune-sorbent method can be used for fluorescence localization and semi-quantitative analysis of ZO-1 for human epithelial colorectal adenocarcinoma cells (Caco-2) and human brain capillary endothelial cells (hCMEC/D3). And the result is identical with results of immunofluorescence and Western blotting based on ZO-1 antibody methods. In the study, a series of APTs for ZO-1 are firstly screened and expected to provide a new tool molecule with high specificity for fluorescent labeling and semi-quantitative analysis of ZO-1.