In order to better control nosocomial infections, and facilitate the most prudent and effective use of antibiotics,improved strategies for the rapid detection and identiﬁcation of problematic bacterial pathogens are required. DNA aptamers have much potential in the development of diagnostic assays and biosensors to address this important healthcare need, but further development of aptamers targeting common pathogens, and the strate- gies used to obtain speciﬁc aptamers are required. Here we demonstrate the application of a quantitative PCR (qPCR) controlled Cell-SELEX process, coupled with downstream secondary-conformation-based aptamer proﬁling. We used this approach to identify and select DNA aptamers targeted against uropathogenic Escherichia coli, for which speciﬁc aptamers are currently lacking, despite the prevalence of these infections. The use of qPCR to monitor the Cell-SELEX process permitted a minimal number of SELEX cycles to be employed, as well as the cycle-by-cycle optimisation of standard PCR ampliﬁcation of recovered aptamer pools at each round. Identiﬁca- tion of useful aptamer candidates was also facilitated by proﬁling of secondary conformations and selection based on putative aptamer secondary structure. One aptamer selected this way (designated EcA5-27), displaying a guanine-quadruplex sequence motif, was shown to have high afﬁnity and speciﬁcity for target cells, and the potential to discriminate between distinct strains of E. coli, highlighting the possibility for development of aptamers selectively recognising pathogenic strains. Overall, the identiﬁed aptamers hold much potential forthe development of rapid diagnostic assays for nosocomial urinary tract infections caused by E. coli.