L-lactate oxidase (LOX) is a biotechnologically important enzyme used in biosensors and colorimetric kits to detect lactate, a key biomarker in clinical diagnostics, sports medicine and the food industry. In this work, we produced a recombinant His-tagged Aerococcus viridans LOX (rLOX) in Escherichia coli and carried out its functional characterization for industrial applications. Our rLOX was evaluated in a colorimetric kit for human diagnostics and in an amperometric biosensor to measure the lactic acid in food products. The rLOX was fully functional for both applications, with a performance comparable to commercial untagged LOXs. As the industrial use of LOX enzyme requires a large-scale production, we scaled up the rLOX production in a fed-batch bioreactor culture and obtained a yield approximately ten times higher than that of the Erlenmeyer scale. The His-tag allowed an easy and highly efficient purification process, and a high-purity rLOX was recovered after this one-step affinity purification. In this study, we described a simple, rapid and cost-competitive approach for the production of a recombinant His-tagged LOX enzyme suitable for industrial use.