Soil microbial community fundamentally drives decomposition process, and thereby plays central roles in nutrient cycling in terrestrial ecosystems. Recent studies have found enormous phylogenic diversity of soil microbes and relationship between decomposition process and microbial community composition. Thus, the evaluation of microbial phylogeny and abundance is important to understand and predict soil decomposition process. Fluorescence in situ hybridization (FISH) coupled with catalyzed reporter deposition (CARD) is a common tool to identify microbial phylogeny and abundance in aquatic systems, but it is seldom applied for soil systems. In this study, we applied CARD-FISH to investigate soil microbial phylogeny and abundance. Application of CARD-FISH for soil samples resulted in the successful detection of different microbial groups. We further developed automated cell counting program by free statistical software R. Simple automated counting of microbial cells was interrupted by autofluorescence of soil particles. To distinguish microbial cells and soil particles, we incorporated some criteria (e.g. circularity, area and fluorescence color) to the program. The enumeration by our improved program agreed well with that by the manual counting. Our method combining CARD-FISH and automated cell counting is cost-effective, and can be applied for any studies in the field of soil microbial ecology.