A new method was proposed for the determination of aflatoxin B1（AFB1） by lateral flow assay based on molecule beacon（MB）/fluorescent microsphere（FM）.A MB containing AFB1 aptamer conjugated with FM,which was coated on the conjugate pad of a strip.A sequence containing adenine（A） was coated on the nitrocellulose membrane（NCM） at the test line,and another sequence complementing with AFB1 aptamer was coated on the control line.In the presence of AFB1,the specific combination of AFB1 and aptamer-FM led to the broken off of the double strand in MB.Then,the complex of AFB1/aptamer-FM was captured at the test line,and the excess MB/FM were captured at the control line.The fluorescence was observed with a fluorescence detector,and the content of AFB1 was estimated by the ratio of fluorescence peaks of the test line（T） and the control line（C）.The results showed that there existed a good linear relationship between the ratio of fluorescence peaks and AFB1 in the range of 0.1-50 μg/L with a limit of detection（S/N=3） approximately 0.05 μg/L.With a light signal “off-on” procedure,this proposed method could provide a new way for the accurate determination of AFB1.