Polymerization of luminol performed in strongly acidic aqueous solution can produce thin electrochemically active films of one redox couple. The film can be produced on glassy carbon, platinum, gold and transparent semiconductor tin(IV) oxide electrodes. An electrochemical quartz crystal microbalance and cyclic voltammetry were used to study in situ growth of the polyluminol film. The modified film is electrocatalytically active for NADH oxidation in acidic and neutral aqueous solutions, and the electrocatalytic oxidation current develops from the anodic peak of the redox couple. The direct electrocatalytic oxidation of epinephrine, dopamine, ascorbic acid, and l-cysteine by the polyluminol film in acidic and neutral aqueous solutions also shows electrocatalytic oxidation activity. The electrocatalytic oxidation properties of dopamine, ascorbic acid, and epinephrine by polyluminol film in the presence of mixtures of the three compounds or of only one compound are also discussed. The results show a selective measurement of dopamine and ascorbic acid. The polymer films were more stable in acidic solutions, their formal potentials being pH-dependent with a slope of −58 mV pH−1.