A novel Fluorescence Resonance Energy Transfer (FRET) based ‘Turn-ON’ biosensor has been developed using fluorescent ZnO/APTMS-FITC (ZFA) nanoflakes as sensing probe. In this biosensor, Lactate Dehydrogenase (LDH) is used for the detection of L-lactate, a diagnostic marker for abnormal physiological conditions like muscular dystrophy, myocardial infraction, abnormal tissue formation and tissue damage. Lactate Dehydrogeanse (LDH) catalyses the conversion of L-Lactate to L-Pyruvate, in presence of β-NAD reducing to β-NADH. We tried to explore this mechanism with FRET based system for highly sensitive detection of L-Lactate. The fluorescence of these nanoflakes can be reversibly quenched in the presence of β-NAD.