Several types of enzymes, including cellulases and xylanases, are required to degrade hemicelluloses and cellulose, which are major components of lignocellulosic biomass. Such degradative processes can be used to produce various useful industrial biomaterials. Screening methods for detecting polysaccharide-degrading microorganisms include the use of dye-labeled substrates in growth medium and culture plate staining techniques. However, the preparation of screening plates, which typically involves chemical cross-linking to synthesize a dye-labeled substrate, is a complicated and time-consuming process. Moreover, such commercial substrates are very expensive, costing tenfold more than the natural xylan. Staining methods are also problematic because they may damage relevant microorganisms and are associated with contamination of colonies of desirable organisms with adjacent unwanted bacteria. In the present study, we describe a sonication method for the simple and rapid preparation of an insoluble substrate that can be used to screen for xylanase-expressing bacteria in microbial populations. Using this new method, we have successfully isolated a novel xylanase gene from a xylolytic microorganism termed Xyl02-KBRB and Xyl14-KBRB in the bovine rumen.