This work used three fungi, Rhizopus oligosorus BCRC 31996, Monascus pilosus BCRC31527, and Aspergillus sp. BCRC31742, to produce glucosamine by using submerged fermentation and flask cultures. The reaction of glucosamine with I-naphthyl isothiocyanate as derivatizing agent was carried out in pyridine at 50 degrees C for 1 h. The derivative was accurately analyzed and quantified by using high performance liquid chromatography. The relative standard deviation of glucosamine determined between experimental and real values were less than 2%. The kinetic and strategy of producing glucosamine in a flask culture was investigated to achieve an optimum yield of glucosamine under different conditions including three kinds of fungi, medium, and pH values. The descending ability of producing glucosamine for the three fungi was Aspergillus sp. BCRC31742 > Monascus pilosus BCRC31527 > Rhizopus oligosorus BCRC 31996 under the conditions studied. The experimental result shows that the glucosamine concentration had an optimum value and was 3430 mg/L by using Aspergillus sp. BCRC31742 culture in glucose and peptone (GP) medium, the yield of which was the best amount using wild-type microorganisms in the past. The generation culture of fungi and the pH control played important roles in enhancing the yield of glucosamine. The specific growth rate of the microorganism and the biomass, content, yield, and productivity of glucosamine were calculated as well.