In this study, a photoelectrochemical glucose biosensor is presented, comprising electropolymerized hematoxylin film on poly-amidoamine (PAMAM) dendrimers adsorbed on glassy carbon electrode (polyHT/PAMAM/GCE). After immobilization of glucose dehydrogenase (GDH) onto the poly-HT/PAMAM/GCE, photoelectrochemical biosensing of glucose was investigated using cyclic voltammetry and amperometry in a flow injection analysis (FIA) system dependent on the NAD+/NADH redox coupledehydrogenase enzyme. The linear range was from 1 x 10(-5) M to 1 x 10(-3) M with a sensitivity of 0.76 mu A mM(-1) and detection limit of 3.0 mM without irradiation in the FIA system. After irradiation, the linear range was from 5 x 10(-6) M to 1 x 10(-3) M with a sensitivity of 1.90 mu A mM(-1) and a detection limit of 1.5 mM. Comparing the results obtained from irradiation of the electrode surface with those from the reaction without irradiation, the sensitivity and the detection limit increased around 2.5- and 2.0-fold, respectively. The photoelectrochemical biosensor showed good performance with high upper detection limit, acceptable repeatability, and selectivity, providing a rapid alternative method for monitoring biomolecules and extending photoelectrochemical determination in the FIA system. The proposed electrochemical and photoelectrochemical biosensor was successfully applied to determination of glucose in real samples. The results obtained from this study provide a basis for future studies in these directions.