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A novel chemiluminogenic reagent, refered to as spiro form fluorescein hydrazide, was synthesized, and its application to chemiluminescence (CL) determination of Co2+ is described. The method is based on the catalytic effect of Co2+ on the autoxidation of sulfite in basic solution to generate a series of powerful oxidative intermediates, which then oxidize colorless, nonfluoresent fluorescein hydrazide to generate strong CL emission. The CL signal is linearly related to the concentration of Co2+ in the range of 0.1–200 nM with a detection limit of 0.04 nM. The optimal conditions for the detection of Co2+ were evaluated, and the possible CL mechanism is discussed. The proposed method has been applied to the determination of Co2+ in pharmaceutical preparations and in soil samples.