Understanding the roles and functions of xylan is crucial for lignocellulose based fuels and materials production, however, tracking xylan in cellulosic substrates has been challenging. In this study, a xylan specific binding domain from thermostable bacteria Thermotoga thermarum DSM 5069 xylanase (Xyn10A) was cloned and characterized as a molecular probe (N1-N2) to monitor the interfacial xylan of cellulosic fibers. The results showed that the N1-N2 could selectively interact with both insoluble and soluble xylanolytic substrates (no affinity with either crystalline or amorphous cellulose). Compared with the well-known thermostable xylan-binding domain from Thermotoga maritima xylanase A, the tandem N1-N2 from Xyn10A exhibited two times higher binding affinity towards xylo-oligosaccharide (xylopentose, 8.8 × 105 M-1), as assessed by Isothermal titration calorimetry. This N1-N2 probe was thermostable at 80oC and could still be functional after 2 h of incubation at 90oC. Visualization of fluorescently labeled N1-N2 by confocal microscopy showed distinguishable distribution of surface xylan from bleached hardwood (BHK, 17.2% xylan) and softwood (BSK, 8.4% xylan) Kraft pulps, respectively. Our results showed that the patterns of interfacial xylan varied greatly among different plant species, and also demonstrated the potential of using thermostable xylan binding domain as a molecular probe to map the location of interfacial xylan during various lignocellulose pretreatment and/or pulping processes.