2,5-Furandicarboxylic acid (FDCA) can be used for synthesis of various polyesters and polyamides. It can be produced from oxidation of 5-hydroxymethylfurfural (HMF), a biomass-based platform chemical. In this work, a new catalyst CotA laccase/TEMPO/catalase has been presented and used for efficient and selective oxidation of HMF to FDCA. Dioxygen O2, which is in-situ generated from the decomposition of H2O2 by catalase, is used as the oxidant. In comparison to using ambient air as the oxidant, using the in-situ generated O2 as the oxidant has significantly increased the catalytic efficiency. Dioxygen O2 can be generated in a convenient and easy way using clean H2O2 as the source. It has been found that continuous generation of O2 is essential, and over generation of O2 is not necessary because of the limitation of the O2 diffusion into the inner space of CotA laccase. It has been demonstrated that, by coupling with the electron transfer mediator TEMPO and catalase, the bacterial laccase can efficiently oxidize HMF to produce FDCA.