A rapid method for the determination of fungal glucosamine (GlcN) from Aspergillus sp BCRC 31742 was developed. The hydrochlorination process using microwave effectively reduced reaction time needed for GlcN analysis. The analytical method consisted of two steps: (1) hydrochlorination of fungal cells and (2) derivatization process. Fungal GlcN hydrochloride was reacted with 1-napthyl isothiocyanate (1-NITC) as the derivatizing agent to enhance the sensitivity of GlcN and so to achieve high resolution. This method was specific for quantification of GlcN hydrochloride at the wavelength of 230 nm. The standard deviation and relative error of the analytical results were less than 5%. By using microwave heating, the reaction time of hydrochlorination process was shortened from 24 h to 3 min. Thus, the overall time needed for analyzing GlcN from fungal sources was reduced from 5 h (thermal method) to 2 h (microwave method).