Escherichia coli o157:h7 is a major food borne bioterrorism relevant pathogen that causes life-threatening infections in humans and other farm animals. to avoid the ill full effects of this pathogen, there is need for development of sensitive and selective bio-ligands for low cost diagnostic assay development. in the present study we aimed to develop ssdna aptamer ligands against target agent using selex (systematic evolution of ligands by exponential enrichment) protocol. to improve the selectivity of the ssdna apta ligands against target pathogen, counter selection was performed using closely related enterobacteriaceae members as well as other bacterial genera. after 8 rounds of the cell-selex process, the selected aptamer (eco1,2 and 3) was found to specifically bind to the o157:h7 serotype, with low or no cross reactivity with other e. coli strains used in present study. moreover, no reactivity was found with the other bacterial strains used in the counter selection. secondary structure of the selected aptamer and dissociation constant (kd) value against target was calculated as 12 nm using m-fold bioinformatics tool. generated ssdna aptamer ligand was further used to develop a sensitive alisa method for specific detection of escherichia coli o157:h7 from contaminated food samples. in conclusion, developed ssdna aptamer may find a potential application in diagnostic development against e. coli o157:h7 induced food borne illness.