A new fluorescent reagent 2,4-dihydroxyacetophenone benzoylhydrazone (DHAPBH) was synthesized and its ionization constants were established spectrophotometrically. The fluorescent reaction of this reagent with cadmium was studied. The cadmium ion can form a stable binary chelate with DHAPBH, having a mole ratio of 1:2 in the pH range of 3.6–4.4. Based on this chelation reaction, a highly sensitive spectrofluorimetric method was developed for determination of trace amounts of cadmium in a water-ethanol (7:3 in volume ratio) medium at pH = 4.0. Under these conditions, the Cd-DHAPBH complex has excitation and emission maxima at 306 and 437 nm, respectively. The linear range of the method is from 0.36 to 140 μg·L −1 and the detection limit is 0.27 μg·L −1 of cadmium. Interferences of other ions were studied. The method was successfully applied to the determination of cadmium in mineral water, tap water, river water, and wastewater.