For fuel cell applications, direct oxidation of L-ascorbic acid was investigated using electrochemical and in situ UV-visible spectroscopic measurements. A typical conductive polymer, polyaniline (PANI), was employed as an electrocatalyst and was electrodeposited on a glassy carbon electrode as a film. The modification of the PANI film significantly increased the anodic current for the oxidation of L-ascorbic acid. The electrocatalytic activity of the PANI film was evaluated for (i) the effect of the type of PANI's dopant anion, (ii) the durability of catalytic action, and (iii) the effect of metal dispersion on the PANI film. The highest electrocatalytic activity was obtained using the SO42--doped PANI film. The durability of the catalytic action was significantly improved by the modification of the PANI film. The metal deposition deactivated rather than activated the PANI film-modified electrode. Both spectroscopic and electrochemical measurements revealed that the electrocatalytic oxidation of L-ascorbic acid occurred through a mediated electron transfer on the PANI film. The PANI film-modified electrode is regarded as a promising anode for direct ascorbic acid fuel cells because it was highly active toward the electro-oxidation of L-ascorbic acid for a long time and was entirely free from harmful heavy metals and precious Pt.