A reliable method, based on the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), was developed for the extraction of viruses from soil and optimized for the detection of Pepper mild mottle virus (PMMoV) in soil taken from green pepper (Capsicum annuum) fields. When added to phosphate buffer, Tween 20 increases extraction efficiency and skim milk increases the specificity for PMMoV. Samples positive by DAS-ELISA were verified by inhibition testing using specific anti-PMMoV antibody, immuno-electron microscopy, reverse transcription-polymerase chain reaction, and inoculation tests on assay plants. Our system for detecting PMMoV in soil was successfully tested on samples from 22 infected and uninfected fields in Japan. When used before seedlings are planted, this method allows for the prediction of possible damage to cultivated plants by soil-borne PMMoV.