Nanomaterials-based colorimetric immunoassays showed increasing attention for monitoring different biomarkers because of their unique optical and electrical features. Here, a highly sensitive and selective optical sensor was described for the determination of different aflatoxins (AFs). Mesoporous silica nanoparticles (m-SNPs) with an average particle size of 40 nm were prepared by the sol-gel method and then decorated with gold nanoparticles (AuNPs). The Au NPs@m-SiNPs nanocomposite with an average particle size of 66 nm was modified with AFs antibodies. The assay includes the following steps: the Au NPs@m-SiNPs nanocomposite was immersed with AFs antibodies, and then the AFs-Ab/Au NPs@m-SiNPs was used as a probe for AFs detection. The interaction between the AFs-Ab/Au NPs@m-SiNPs and the AFs has resulted in a change in its color from pink to violet. Measurements are performed by absorptiometry at a wavelength of 425 nm. The immunoassay works in the concentration range from 1 ng·mL to 75 ng·mL AFB1 and has a limit of detection 0.16 ng·mL (at S/N = 3). The assay was applied to the determination of AFs in different food samples spiked with AFS. Finally, the assay was used to detect AFs in a real sample, and the LC-MS technique was used to verify the results.