Bio-based 2,5-furandicarboxylic acid (FDCA) has the potential to replace petroleum-based terephthalic acid for the synthesis of high-performance polyester materials, demonstrating broad application prospects. Its primary preparation method involves the selective oxidation of 5-hydroxymethylfurfural (HMF). However, the industrial process is limited by low HMF concentration during the reaction due to the formation of humus resulting from HMF instability especially in high concentration. In this study, a RuCo/NC bimetallic catalyst was fabricated, which can effectively catalyze the selective oxidation of HMF to obtain FDCA at room temperature (25 °C). Side reactions caused by HMF instability were significantly reduced at room temperature, allowing for the application of high-concentration HMF (10 wt%) to achieve an excellent FDCA yield of 91.92% in water. Mechanism studies reveal that a synergistic electronic effect exists between two metals that electrons transfer from Co to Ru to increase the electron density on the surface of Ru nanoparticles, improving oxygen activation ability. Meanwhile, the electron-deficient Co further enhances the adsorption of HMF on the catalyst surface for better reactivity. This study realized the high-concentration HMF aerobic oxidation to FDCA at room temperature in water, paving the way for FDCA to serve as a sustainable substitute for terephthalic acid in polyester production.