Antibody-based technology is the main method for diagnosis and treatment of snake bite envenoming currently. However, the development of an antibody, polyclonal or monoclonal, is a complicated and costly procedure. Aptamers are single stranded oligonucleotides that recognize specific targets such as proteins and have shown great potential over the years as diagnostic and therapeutic agents. In contrast to antibodies, aptamers can be selected in vitro without immunization of animals, and synthesized chemically with extreme accuracy, low cost and high degree of purity. In this study we firstly report on the identification of DNA aptamers that bind to β-bungarotoxin (β-BuTx), a neurotoxin from the venom of Bungarus multicinctus. A plate-SELEX method was used for the selection of β-BuTx specific aptamers. After 10 rounds of selection, four aptamer candidates were obtained, with the dissociation constant ranged from 65.9 nM to 995 nM measured by fluorescence spectroscopy. Competitive binding assays using both the fluorescently labeled and unlabeled aptamers revealed that the four aptamers bound to the same binding site of β-BuTx. The best binder, βB-1, bound specifically to β-BuTx, but not to BSA, casein or α-Bungarotoxin. Moreover, electrophoretic mobility shift assay and enzyme-linked aptamer assay demonstrated that βB-1 could discriminate B. multicinctus venom from other snake venoms tested. The results suggest that aptamer βB-1 can serve as a useful tool for the design and development of drugs and diagnostic tests for β-BuTx poisoning and B. multicinctus bites.