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Fast and sensitive detection of E.coli O157: H7 is significantly essential for clinical management as well as for transmission prevention during disease outbreaks. Though many types of detection strategies have been implemented for measuring E.coli O157: H7, most of them still rely on complex instruments or tedious/laborious setups, which restrict their applications in resource-limited scenarios. Herein, we introduce an eye-based microfluidic aptasensor (EA-Sensor) for fast detection of E.coli O157: H7 without the assist of any instruments. We demonstrate the perfect coupling of aptamer sensing, hybridization chain reaction (HCR)-amplification and a distance-based visualized readout to quantitatively determine the pathogen concentration. We first used gel-electrophoresis assay to evaluate the system and the results proved that E.coli O157: H7 was well recognized by the aptamer and HCR could increase the signal by about 100 folds. In addition, the Aptamer specificity and signal-amplification ability were verified on the EA-Sensor for sensing E.coli O157: H7 by naked eyes. Furthermore, we demonstrated that E.coli O157: H7 in milk could be accurately and conveniently measured with good performance. With the benefits of operation integration and strategy integration, our EA-Sensor shows advantages of high specificity, easy operation, efficient amplification and visualized readout, which offers a favorable point-of-care tool for E.coli O157: H7 or other pathogen detection in resource-constrained settings. Image 1 • An eye-based aptasensor (EA-Sensor) is developed for point-of-care detection of E.coli O157:H7. • Integration of aptamer recognition, HCR amplification and a visualized readout is firstly realized for pathogen detection. • On-chip measurement of E.coli O157:H7 in milk samples was successfully achieved. [ABSTRACT FROM AUTHOR]