In this study, a novel amphoteric macromolecule was synthesized by sequential incorporation of carboxymethyl and quaternary ammonium groups into the backbone of xylan-type hemicelluloses under microwave irradiation. The reaction parameters such as the molar ratio of reagent (NaOH or 3-epoxypropyltrimethylammonium chloride)/anhydroxylose unit in hemicelluloses, the temperature, and the reaction time were investigated to optimize the reaction condition. The maximum degrees of substitution (DS) of carboxymethyl and quaternary ammonium groups under the optimum reaction condition were 0.90 and 0.52, respectively, exhibiting a higher efficiency as compared to the conventional heating method. Moreover, the thermal stability and weight-average molecular weight of amphoteric hemicellulosic derivatives decreased as compared to the native hemicelluloses. The viscosity, elastic modulus, and loss modulus of the amphoteric biomacromolecules increased with the increasing DS of quaternary ammonium groups in aqueous solution due to stronger electrostatic attraction. This study provides an efficient and rapid method to prepare amphoteric biomacromolecules.