Aflatoxins (AFs) are carcinogenic and toxic secondary metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus. To monitor and regulate the AF contamination of crops, a sensitive and precise detection method for these toxigenic fungi in environments is necessary. We herein developed a novel visual detection method, the dichlorvos-ammonia (DV-AM) method, for identifying AF-producing fungi using DV and AM vapor on agar culture plates, in which DV inhibits the esterase in AF biosynthesis, causing the accumulation of anthraquinone precursors (versiconal hemiacetal acetate and versiconol acetate) of AFs in mycelia on the agar plate, followed by a change in the color of the colonies from light yellow to brilliant purple-red by the AM vapor treatment. We also investigated the appropriate culture conditions to increase the color intensity. It should be noted that other species producing the same precursors of AFs such as Aspergillus nidulans and Aspergillus versicolor could be discriminated from the Aspergillus section Flavi based on the differences of their phenotypes. The DV-AM method was also useful for the isolation of nonaflatoxigenic fungi showing no color change, for screening microorganisms that inhibit the AF production by fungi, and for the characterization of the fungi infecting corn kernels. Thus, the DV-AM method can provide a highly sensitive and visible indicator for the detection of aflatoxigenic fungi.