In this study, a new method is proposed for the determination of trace amounts of folic acid (vitamin B₉). This method is based on the inhibitory effect of folic acid on the reaction of Thionine and bromate in sulfuric acid media. The reaction can be monitored spectrophotometrically by measuring the decrease in absorbance at 601nm (λ_max). The effective variables on the reaction rate were investigated. Under optimum experimental conditions, the method allows to determine of the folic acid in a wide linear range with two linear segments. The limit of detection was 0.36μgmL⁻¹ of folic acid. Relative standard deviations of six replicate determinations of 5.0 and 50.0μgmL⁻¹ of folic acid were 1.18 and 1.02%, respectively. The interfering effect of the different species was also investigated. The method was evaluated by quantifying of folic acid in biological and pharmaceutical samples with satisfactory assay results.