Aflatoxin B1 (AFB1) is the most toxic mycotoxin, which is classified as grade 1 carcinogens by the World Health Organization. A method for detection of AFB1 in moldy peanut was proposed by using lysinefunctionalized gold nanoparticles (Lys-AuNPs). AuNPs was synthesized by the citrate reduction method, of which the color was redwine. To obtain lysinefunctionalized AuNPs, lysine solution was added to the prepared AuNPs under stirring by magnetic stirrer. It was characterized by transmission electron microscope and UVvisible spectrophotometry. The detection of AFB1 was achieved through the competitive reaction mechanism between Hg2+, AFB1 and amino acids on lysine surface in the gold nanoparticles solution. As the concentration of AFB1 in Lys-AuNPs solution was increased, the color of the solution was changed from grey to red. UVvisible spectrums were measured to establish the relationship between the A725/A525 value of Lys-AuNPs solution and the concentration of AFB1. The results showed that there was a good linear relationship in the range of 1~50ng/mL, and the correlation coefficient was 0996. The detection limit of AFB1 was 0.2ng/mL and the recovery in peanut samples was 85%~110%. In addition, the concentration of AFB1 in peanut was confirmed by high performance liquid chromatography (HPLC). The method for detection of AFB1 in moldy peanuts had the advantages of quick simplicity, high sensitivity, and simple operation and so on, which can be applied to the rapid detection of aflatoxin B1 in moldy peanut and make the assay widely applicable.