A novel dual-competitive lateral flow aptasensor (LFA) benefited from aptamer and lateral flow strips was first designed by using aflatoxin B-1 (AFB(1)) as the model target. In this LFA assay, the target AFB(1) competed with AFB(1)-hapten at T line for binding to Cy5-labeled AFB(1) aptamer and the complementary strand competed with the target AFBi for binding to Cy5-labeled AFBi aptamer at C line. The ratio of their fluorescent intensities at the T line and C line (ST/SC ratio) was employed in order to increase the sensitivity for target AFB(1) detection. This technique has the limit of detection (LOD) of 0.1 ng/mL for AFB(1) within the linear range from 0.1 ng/mL to 1000 ng/mL. Subsequently, the LFA approach was validated using 11 kinds of food and feedstuff samples with a simple aqueous extraction protocol. The test results with different naturally contaminated feedstuffs indicated a good correlation between this LFA and a commercial ELISA kit. The assay can be completed within 20 min and its sensitivity, specificity and reproducibility are highly satisfactory. This is the first LFA that has been rigorously validated, which will be greatly beneficial to development of commercial aptamer-based biosensors for food safety, environmental analysis, particularly in clinical diagnosis. (C) 2017 Elsevier B.V. All rights reserved.