Diabetic retinopathy (DR) is the leading global cause of blindness in the working-age population. Early diagnosis and intervention can effectively reduce the risk for blindness. However, the current diagnostic methods in clinical practice remain constrained by nonquantitative examinations and individual ophthalmologists’ experiences. Sensitive, specific and accurate detection of DR-specific biomarkers is an important approach to achieve its early and rapid diagnosis. In this study, a high-affinity aptamer APT12TM that specifically binds to the tear-derived DR biomarker lipocalin 1 was obtained. The aptamer APT12TM can be folded into a stable B-DNA structure, and its strong interaction with LCN 1, including hydrogen bonding and hydrophobic interactions, is an important factor for targeted recognition and high-affinity binding. A G-rich DNA fragment was further assembled at both ends of the aptamer APT12TM, and the B-DNA form was successfully converted into a parallel G-quadruplex. Most importantly, LCN 1 could induce further transformation of the G-quadruplex structure. Therefore, a fluorescent aptasensor based on G-quadruplex-assisted structural transformation was developed through the Thioflavin T mediator. The aptasensor exhibited a broad detection window from 0.25 to 1000 nM LCN 1, with a limit of detection of 0.2 nM. Furthermore, the aptasensor was applied to LCN 1 detection in artificial tear samples and displayed good reproducibility and stability. These results show that the developed aptasensor has significant potential for sensitive, specific and convenient detection of the DR-specific biomarker LCN 1.Copyright © 2020 Elsevier B.V. All rights reserved.