2,5-Furandicarboxylic acid (FDCA) is an important monomer for manufacturing biobased plastics. Biocatalysis has been recognized as a sustainable tool in organic synthesis. To date, the efficiencies of most biocatalytic processes toward FDCA remain low. So, it is highly desired to develop efficient processes. In this work, a biocatalytic route toward FDCA was developed by integrating a cell-free extract of galactose oxidase variant M3−5 with a whole-cell biocatalyst harboring NAD+-dependent vanillin dehydrogenases and NADH oxidase, starting from 5-hydroxymethylfurfural. FDCA was produced in a concurrent mode with >90% yields within 36 h at 20 mM substrate concentration. In addition, biocatalytic synthesis of FDCA was performed on a preparative scale, with 78% isolated yield. The present work may lay the foundation for sustainable production of FDCA.