To design the Aspergillus flavus and Aspergillus parasiticus-specific primers and a real-time PCR assay for quantification of the conidial density in soil. Aspergillus flavus and A. parasiticus-specific DNA primers were designed based on internal transcribed spacer sequences to distinguish these two species and from other Aspergillus and other fungal species. A method of pathogen DNA extraction directly from soil samples was developed. Using the designed primers, a real-time PCR assay was developed to quantitatively determine the conidial density of each A. flavus and A. parasiticus in soil, after generating corresponding standard curves. Known conidial densities of each A. flavus or A. parasiticus in soil significantly correlated with those tested with the real-time PCR. This study demonstrated the applicability of the real-time PCR assay in studies of quantifying A. flavus and A. parasiticus in soil as inoculum sources. The A. flacus and A. parasitic-specific primers can be widely used in aflatoxin research. The real-time PCR assay developed in this study provides a potential approach to quantify the plant pathogen density from not only soil but also other sources in relation to aflatoxin contamination from environment, food and feed commodities.