Understanding the roles and functions of xylan is crucial for lignocellulose-based fuel and material production; however, the tracking of xylan in cellulosic substrates has been challenging. In this study, a xylan specific binding domain from the thermostable Thermotoga thermarum DSM 5069 xylanase (Xyn10A) N-terminal domain (N1–N2) was cloned and characterized as a potential molecular probe 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). The N1–N2 probe was thermostable at 80 °C and still maintained the high xylan-binding affinity even after 2 h of incubation at 90 °C. 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. These differences were similar to the ...