The invention discloses a nucleic acid aptamer test strip for detecting aflatoxin B1 as well as a preparation method and an application of the nucleic acid aptamer test strip. The test strip comprisesa sample pad, a nitrocellulose membrane, a water absorption pad, a supporting layer and a matching reagent, wherein the matching reagent is a nano-gold labeled aflatoxin B1 aptamer solution; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a biotinylated probe 1-streptavidin solution, and the quality control line is coatedwith a biotinylated probe 2-streptavidin solution. The test strip has the advantages of being high in sensitivity and specificity, simple to operate, economic, practical and the like, and is suitablefor rapid detection of aflatoxin B1 residues.