2, 5-furandicarboxylic acid (FDCA) is a one of the most promising biomass-derived chemicals to substitute the non-renewable terephthalic acid as the monomer for producing polyethyleneterephthalate. At present, the oxidation of HMF is regarded as a prevalent way to prepare FDCA. Nevertheless, the isolation and storage of HMF is still a challenge. Herein, based on the higher stability of 5-formyloxymethylfurfural (FMF) than 5-hydroxymethylfurfural (HMF), we present an effective preparation route to prepare FDCA by substituting HMF with FMF as feedstock. A complete conversion of FMF and a 93.55% selectivity of FDCA were obtained in the mixed solvent of water and 1, 2- dioxane using Ru/C as catalyst and O2 as oxidant. An improved process was developed for preparing FDCA using FMF as feedstock. The investigation of conversion pathway showed that FMF and HMF were simultaneously oxidized to 2, 5-diformylfuran (DFF) in a case of the existence of the reversible equilibrium between FMF and HMF. Then DFF was oxidized to 5-formyl-2-furancarboxylic acid (FFCA). Subsequently, FFCA was oxidized to FDCA. In this process, the oxidation of FFCA to FDCA was determined as the rate-determining step. Furthermore, appropriate alkalinity favored the selectivity of FDCA and the conversion of FMF.