beta-Xylosidase is a key enzyme in the xylanolytic system with a great potential in many biotechnological applications, especially in the food as well as the pulp and paper industries. We have developed a chemical approach for the rapid screening and isolation of beta-xylosidase. Activity probe LCL-6X targeting beta-xylosidase was utilized in this study. It carries a beta-xylopyranosyl recognition head, a latent trapping device consisting of a 2-fluoromethylphenoxyl group, and a biotin reporter group. The biotin reporter group serves both as a readout device and as a tool for enriching the labeled proteins. LCL-6X could selectively label a model beta-xylosidase from Trichoderma koningii. All other bystander proteins used in this study, including phosphorylase b, BSA, ovalbumin, carbonic anhydrase, and trypsin inhibitor, gave negligible cross-labeling effect. With the assistance of streptavidin agarose beads and mass spectrophotometry for the recovery and identification of the biotinylated proteins, we demonstrated that LCL-6X could be successfully applied to identify a bi-functional enzyme with alpha-L-arabinofuranosidase/beta-xylosidase activity from the total protein extract of a Pichia expressing system and a prospective beta-xylosidase in the culture medium of Aspergillus fumigatus. The beta-xylosidase activities from numerous microbes were also screened using the LCL-6X probe. Preliminary results showed significant differences among these microbial sources and some distinct protein bands were observed. Thus, we have successfully developed a novel chemical probe that has potential applications in xylan-related research.