A photon counting method based on the detection of bioluminescence was developed for the determination of ultratrace levels of β-nicotineamide adenine dinucleotide, reduced form (NADH). A commercial bacterial luciferase (Vibrio fischeri) which contains both flavin mononucleotide (FMN) reductase and luciferase was immobilized by the diazocoupling method onto arylamine glass beads. The immobilized luciferase beads were packed in a tube and placcd in front of the photomultiplier tube of a photon counter. A flow injection system with a packed-bed reactor was used for the determination of NADH. The immobilized enzyme system is superior to soluble luciferase in that it is reusable and much more stable. In the present method. the lower limit of detection of NADH was 130 fmol. Ethanol and L-lactic acid determinations in serum were carried out by using immobilized alcohol dehydrogenase and lactic dehydrogenase, respectively. NADH produced in each enzyme reaction was assayed by using the immobilized luciferase column. The serum background emission is negligibly small. The methods are simple and suitable for routine use.