The present work describes oxidation of ascorbic acid (AA) at octacyanomolybdate-doped-glutaraldehyde-crosslinked poly-l-lysine (PLL-GA-Mo(CN) 4À 8 Þ film modified glassy carbon electrode in 0.1 M H 2 SO 4 . The modified electrode has been successfully prepared by means of electrostatically trapping Mo(CN) 4À 8 mediator in the cationic film of glutaraldehyde-cross-linked poly-l-lysine. The dependence of peak current of modified electrode in pure supporting indicates that the charge transfer in the film was a mixed process at low scan rates (5 to 200 mV s À1 ), and kinetically restrained at higher scan rates (200 to 1000 mV s À1 ). Cyclic voltammetry and rotating disk electrode (RDE) techniques are used to investigate the electrocatalytic oxidation of ascorbic acid and compared with its oxidation at bare and undoped PLL-GA film coated electrodes. The rate constant of catalytic reaction k obtained from RDE analysis was found to be 9.5 Â 10 5 cm 3 mol À1 s À1 . The analytical determination of ascorbic acid has been carried out using RDE technique over the physiological interest of ascorbic acid concentrations with a sensitivity of 75 mA mM À1 . Amperometric estimation of AA in stirred solution shows a sensitivity of 15 mA mM À1 over the linear concentration range between 50 and 1200 mM. Interestingly, PLL-GA-Mo(CN) 4À 8 modified electrode facilitated the oxidation of ascorbic acid but not responded to other electroactive biomolecules such as dopamine, uric acid, NADH, glucose. This unique feature of PLL-GA-Mo(CN) 4À 8 modified electrode allowed for the development of a highly selective method for the determination of ascorbic acid in the presence of interferents.