Isomerization of glucose derived from lignocellulosic biomass is an important step in biorefinery. Fructose isomerized from glucose, is used as a highly attractive sweetener in the food and beverages industries. However, the prevalence of side reactions at high glucose concentrations is a serious issue, leading to a significant reduction in the fructose yield, especially in the aqueous phase. In this study, an efficient method for the conversion of highly concentrated glucose into fructose under low temperature conditions using triethylamine as the catalyst was developed. It was demonstrated that high fructose yield could be maintained at high glucose concentration. At 60℃, fructose yield of 38.7% and fructose selectivity of 80.6% were achieved in 1 mol/L (approximately 17 wt%) glucose. When glucose concentration was increased to 2 mol/L (approximately 31 wt%), the fructose yield and selectivity were maintained at 34.7% and 77.4%, respectively. ¹³C nuclear magnetic resonance (NMR) spectrometer was used to examine the glucose isomerization reaction. Compared to the NaOH catalytic system, triethylamine acted as a buffer to provide a stable alkaline environment for the catalytic system, further maintaining a high level of catalytic efficiency for the isomerization of glucose to fructose.