Aflatoxins (AFs) are a family of fungal toxins that produced in food and feed by two Aspergillus species (Aspergillus flavus and Aspergillus parasiticus). Several techniques have been reported for AFs detection including high-pressure liquid chromatography, enzyme-linked immunosorbent assay, surface plasmon resonance and recombinant immune blotting assay. But, these methods are disadvantaged because they consumed a long time for analysis; in addition, they required a piece of complicated and expensive equipment. Therefore, developing of inexpensive sensors with high selectivity and sensitivity for detecting of AFs levels without extensive sample preparation has received great attention. Several electrochemical AFs sensors have been reported; however, there is still a need for developing a new, simple and rapid electrochemical AFs sensor. Here, we have developed a new AFs sensor based on Au nanostructures/graphene nanosheets modified ITO substrate that could enhance the Raman effect and the electrochemical conductivity. The modified electrode was prepared based on layer-by-layer electrochemical deposition method. AFs antibody was immobilized onto the Au nanostructures/graphene nanosheets; then it was used as a probe for rapid, simple and cheap detection of AFs level using Raman spectroscopy and electrochemical techniques. Our results demonstrated that the developed system showed a simple, easy and sensitive sensor for monitoring low concentrations of AFB1 with a detection limit of about 6.9 pg/mL, also it allowed the determination of AFB1 in spiked food samples.