We developed a new biosensor for the detection of aflatoxin B1(AFB1) based on the interaction of gold nanoparticles (AuNPs) with the aptamer. Aggregation of AuNPs was induced by desorption of the AFB1 binding aptamer from the surface of AuNPs as a result of the aptamer target interaction leading to the color change of AuNPs from red to purple. The linear range of the colorimetric aptasensor covered a large variation of AFB1 concentrations from 80 to 270 nM and the detection limit of 7 nM was obtained. Also, the catalytic activity of the aggregated AuNPs greatly enhanced the chemiluminescence (CL) reaction, where the detection limit was determined at 0.5 nM with a regression coefficient of R(2) = 0.9921. We have also shown that the sensitivity of detection was increased by employing CL and using the catalytic activity of aggregated AuNPs, during luminol-hydrogen peroxide reaction. Therefore the proposed nanobiosensor was demonstrated to be sensitive, selective, and simple, introducing a viable alternative for rapid screening of toxin in foods.