Rhodamine B piperazinoacetohydrazine (RBPH) is used as a bifunctional probe for the N-terminal specific modification of a thermophilic enzyme (T. lanuginosus xylanase), and the modification effect on the thermostability of the enzyme is investigated. The probe RBPH, bearing a spectroscopic unit of rhodamine B, a carbonyl-specific labeling unit of hydrazine and a linker of piperazine, not only has a stable always-on spectroscopic response, but also exists in a cationic form. These properties enable RBPH to serve as a bifunctional probe (simultaneous introduction of stable spectroscopic signal and positive charge) for the protein modification, and such an application has been successfully demonstrated on the N-terminal labeling of T. lanuginosus xylanase. A temperature-dependent inactivation study shows that the modification of T. lanuginosus xylanase by RBPH hardly changes its thermostability, in other words, a small change in electric charge of the N-terminal region caused by introducing one positive charge is not enough to alter the thermostability of the enzyme. This reveals a conservative property of the N-terminal domain for electric charge change, and such a property may result from the fact that the N-terminal domain of the enzyme already has 4 charged residues, which can produce strong electrostatic interactions, thereby making the domain quite stable.