A simple and sensitive spectrofluorometric method was developed for the simultaneous determination of ascorbic acid and cysteine by a flow-injection system. This method is based on the reduction of Tl(III) with ascorbic acid or cysteine in acidic media, producing fluorescence reagent, TlCl₃^2- (λ_ex = 227 nm, λ_em = 419 nm). The injected sample solution was divided into two separate streams. The first stream was treated with Tl(III) at pH 3.0 and then passed through a 270 cm reaction coil to the flow cell of the spectrofluorometer, where the fluorescence intensity was measured. This signal is related to ascorbic acid and cysteine concentration. The second part of the injected sample solution was treated with Tl(III) in HCl solution and then passed through a 50 cm reaction coil to the flow cell and the fluorescence intensity was measured. This signal is related only to cysteine. Thus, the ascorbic acid content was determined directly by the difference according to the calibration curve. Ascorbic acid and cysteine can be determined in the range of 1 × 10^-6 to 5.0 × 10^-5 M, at a rate of 16 samples per hour. The limits of detection (S/N = 3) were 8 × 10^-7 M for ascorbic acid and 7 × 10^-7 M for cysteine. The influence of potential interfering substances was studied. The proposed method was successfully applied to the simultaneous determination of both analytes in real samples.