The present work describes preparation, characterization, and electrocatalytic behavior of a hexacyanoferrate-dopedglutaraldehyde-cross-linked poly-l-lysine (PLL-GA-Fe(CN) 4À 6 Þ film modified glassy carbon electrode. The modified electrode has been successfully prepared by electrostatically binding negatively charged Fe(CN) 4À 6 mediator into cross-linked poly-l-lysine cationic film. The dependence of the peak current of the modified electrode in pure supporting electrolyte (pH 6.8 phosphate buffer solution; PBS) shows that the charge transport in the film is fast and relatively unimpeded at lower scan rates. Cyclic voltammetry and rotating disk electrode (RDE) techniques are used to investigate the electrocatalytic activity of modified electrode towards oxidation of ascorbic acid. The rate constant (k), of catalytic reaction between electrogenerated Fe(CN) 3À 6 ions and ascorbic acid, obtained from RDE analysis was found to be 5.53 Â 10 5 cm 3 mol À1 s
À1. Finally, the PLL-GA-Fe(CN) 4À 6 film electrodes are successfully used for the individual estimation of ascorbic acid in the concentration range of physiological interest.