Enzymatic oxidative dehydrogenation of biomass-derived c-lactate in the presence of O-2 is regarded as a green alternative for pyruvate production, but the process also results in H2O2 accumulation. To remove the effect of H2O2 on pyruvate production, various potential catalases from different species were screened for their efficiency in H2O2 degradation. Then, an in vitro cascade biocatalysis was designed using lactate oxidase from Aerococcus viridans (AvLOX) and catalase from Ureibacillus thermosphaericus (UtCAT). The in situ removal of H2O2 by UtCAT significantly improved the yield and productivity of pyruvate. To simplify the technological processes and reduce production costs, a whole cell biocatalyst without the addition of catalase for the production of pyruvate was established by coexpressing AvLOX and UtCAT in Escherichia coli. By applying suitable coexpression strategies and changing the ribosome binding site (RBS) strengths, the optimal ratio of AvLOX:UtCAT(12.6:4127.3) in E. coli-AvLOX-((rb)(s3)())UtCAT was finally achieved. Under the optimized transformation conditions, pyruvate was produced at a titer of 59.9 g/L and a yield of 90.8% in a substrate fed-batch process, promising an alternative route for the green production of pyruvate.