Soil contains low amounts of protein but high amounts of interfering substances. Current extraction methods for soil protein cannot produce high-quality samples suitable for proteomic analysis. To resolve the problem, we devised a sequential extraction method, through sequentially extracting soil in citrate and SDS buffers, followed by phenol extraction. The method allows for obtaining applicable 1-D and 2-D protein profiles with various agricultural soils and detecting glomalin-related soil protein. The method may be a valuable tool for soil proteomics.