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Based on the chemiluminescence (CL) phenomena of folic acid in peroxomonosulfate-cobalt(II) system, a rapid and sensitive CL method was developed for determination of folic acid in pharmaceutical preparations and its urinary metabolism processes. Under the optimum conditions, the relative CL intensity was linear over the concentration ranging from 10 −9 to 8 × 10 −7 mol L −1 (R 2 = 0.9991) with a detection limit as low as 6 × 10 −10 mol L −1 (S/N = 3) and relative standard deviation was 2.63% for 2 × 10 −8 mol L −1 folic acid (n = 11). This method has been successfully applied to the determination of folic acid in tablets and human urine. The blank CL emission was yielded owing to the formation of singlet oxygen molecular pair from the quenching experiment of 1,4-diazabicyclo[2.2.2]octane, and pterine-6-carboxylic acid might be the degradation intermediate in this system and it also acts an energy acceptor and sensitizes the chemiluminescence based on the studies of the CL and fluorescence spectra.