Biomass hydrolysate, rich in glucose, was used to produce an important platform chemical, 5-hydroxymethylfurfural (HMF). By separating the solid biomass from solution after autohydrolysis, most of the inhibitors were removed from hydrolysate. Biphasic system, which prevents the HMF degradation, was optimized with HCl and AICl(3) catalysts. The yield of HMF conversion using biomass hydrolyzate under the optimized reaction conditions is comparable to the yield using pure glucose as a feedstock. This lab-generated HMF was purified via activated charcoal and oxidized to high value-added chemical, 2,5-furandicarboxylic acid (FDCA). The final FDCA yield of 65% was achieved. The results suggest that, with the separation of nonsugar components such as dissolved lignin and sugar degradation products, biomass hydrolysate is a promising source for HMF and FDCA production.