Large-scale orthorhombic single-crystalline molybdenum trioxide nanowires were synthesized using a facile one-pot hydrothermal method. Lactate oxidase enzyme was immobilized on the nanowires to produce a highly sensitive electrochemical biosensor for L-lactate detection. At an applied potential of 0.5 V, the sensor exhibited a high sensitivity of 0.87 μA/mM with a fast response to L-lactate (90% of response times within 10 s). A linear response was obtained over a concentration range from 0.5 to 8 mM with a detection limit of 0.15 mM (S/N03). The developed biosensor showed excellent reproducibility and operational stability, as well as the ability to be stored long term.