Esophageal cancer, especially esophageal squamous cell carcinoma (ESCC), poses a serious threat to human health. It is urgently needed to develop recognition tools and discover molecular targets for early diagnosis and targeted therapy of esophageal cancer. Here, we developed several DNA aptamers that can bind to ESCC KYSE410 cells with a nanomolar range of dissociation constants by using cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX). The selected A2 aptamer is found to strongly bind with multiple cancer cells, including several ESCC cell lines. Tissue imaging displayed that the A2 aptamer can specifically recognize clinical ESCC tissues but not the adjacent tissues. Moreover, we identified integrin β1 as the binding target of A2 through pull-down and RNA interference assays. Meanwhile, molecular docking and mutation assays suggested that A2 probably binds to integrin β1 through the nucleotides of DA16-DG21, and competitive binding and structural alignment assays indicated that A2 shares the overlapped binding sites with laminin and arginine-glycine-aspartate ligands. Furthermore, we engineered A2-induced targeted therapy for ESCC. By constructing A2-tethered DNA nanoassemblies carrying multiple doxorubicin (Dox) molecules as antitumor agents, inhibition of tumor cell growth in vitro and in vivo was achieved. This work provides a useful targeting tool and a potential molecular target for cancer diagnosis and targeted therapy and is helpful for understanding the integrin mechanism and developing integrin inhibitors.