An approach is developed to detect the organophosphorus pesticides via competitivebinding to a recombinant broad-specificity DNA aptamer with a molecular beacon(MB),the binding of the MB to the aptamer results in the activation of a fluorescentsignal,which can be measured for pesticide quantification.Aptamers selected via theSystematic Evolution of Ligands by Exponential Enrichment(SELEX)werestructurally modified and truncated to narrow down the binding region of the target,which indicated that loops of the aptamer contributed different functions for differentchemical recognition.Thereafter,a variant fused by two different minimum functionalstructures,was clarified with broad specificity and increased affinity.Furthermolecular docking and molecular dynamics simulations was conducted to understandthe molecular interaction between DNA structure and chemicals.3D modelingrevealed a hot spot area formed by 3 binding sites,forces including hydrogen bondsand vander Waals interactions appear to play a significant role in enabling and stabilizing the binding of chemicals.Finally,an engineered aptamer based approachfor the detection of organophosphorus pesticides was successfully applied in a testusing a real sample,the limit of quantification(LOQ)for phorate,profenofos,isocarbophos,and omethoate reached 19.2,13.4,17.2,and 23.4 nM(0.005 mg L-1),respectively.