The present work encompasses a novel combined approach of electrostatic functionalization and thermally embedding of graphene oxide (GO) on a flexible substrate to fabricate a highly sensitive and selective amperometric lactate biosensor. GO was functionalized by non-chemical and dry-process using corona discharge tube method, i.e., electrostatic approach to yield oxygenated functional groups on their surfaces and transferred onto flexible substrate by thermal embedding technique, which acts as a working electrode. Commercial, lactate oxidase from Pediococcus species has been immobilized through glutaraldehyde coupling onto the surface of working electrode. The electrocatalytic activity of the modified electrodes towards lactic acid was investigated using amperometric technique. The enzyme electrode showed good electrocatalytic activity and optimum response when operated at 35oC in 0.05M sodium phosphate buffer at 0.4 V. A linear response with the lactic acid concentration range from 0.1 to 6mM/L and a high sensitivity of 0.1202 μA/mM/l with a detection limit of 0.1mM/L was observed. The serum values in healthy and diseased persons were in the range of 0.5-2.4 and 4.4mM to 8mM, respectively. The analytical recovery of added lactic acid was 84%. The biosensor sustained its stability for more than 25 days with 150 times of reusage without considerable loss of activity, when stored at 40C. The electrode significantly provides long term stability and highly reproducible detection of a key metabolite lactate in serum.