An on-line photochemical spectrofluorimetric method for the determination of nicotinamide is proposed. It is based on the conversion of nicotinamide in an acidic medium into an intensively fluorescent compound. The calibration graph was linear up to 10.0 μg ml−1nicotinamide (r = 0.999), the limit of detection was 0.60 ng ml−1nicotinamide and the relative standard deviations were 1.5% (n = 6), 2.1% (n = 6) and 2.1% (n = 6) for 50 ng ml−1, 0.70 μg ml−1and 1.0 μg ml−1nicotinamide, respectively. The kinetic behaviors of the reaction and the effects of some experimental conditions were investigated. Its application to assay of vitamin B complex tablets was found to be satisfactory.