A stable electroactive thin film of poly-hematoxylin (poly-HT) was successfully prepared on a glassy carbon electrode (GCE) surface by recording successive cyclic voltammograms of 0.3 mM HT, in a phosphate buffer solution (pH 7.0) containing 0.1 M NaNO3, in the potential range of -0.5 to +2.0 V vs. Ag/AgCl. The deposition of HT on GCE surface can be explained through the electropolymerization process. This poly-HT modified electrode exhibited a good electrocatalytic activity towards the NADH oxidation in a phosphate buffer solution (pH 7.0), and led to a significant decrease in the overpotential by more than 320 mV compared with the bare GCE. In order to perform the photoelectrocatalytic determination of NADH in a flow injection analysis (FIA) system, a home-made flow electrochemical cell with a suitable transparent window for irradiation of the electrode surface was constructed. Flow rate of carrier solution, transmission tubing length, injection volume and applied potential for the amperometric and photoamperometric FIA studies were optimized as 1.3 mL min(-1),10 cm, 100 mu L and +300 mV vs. Ag/AgCl, respectively. The currents obtained from amperometric and photoamperometric measurements in FIA system at optimum conditions were linearly dependent on the NADH concentration and linear calibration curves were obtained in the range of 1.0 X 10(-7)-1.5 X 10(-4) M and in the range of 1.0 x 10(-7)-2.5 X 10(-4) M NADH, respectively. The relative standard deviation (RH)) of six replicate injections of 6.0 x 10(-5) M NADH was calculated as 2.2% and 4.3% for the amperometric and the photoamperometric method, respectively. The limit of detection was found to be 3.0 x 10(-8) M for the photoamperometric determination of NADH. (C) 2010 Elsevier B.V. All rights reserved.