Abstarct: In this study, a flow injection method was described for determination of L-Cysteine in water, humen serum and urine. The method is baxsed on the inhibition effect of L-Cysteine on the reaction between mexta Cresol Purple (MCP) and potassium bromate that catalyzed by bromide ions. In the presence of L-Cysteine an induction period was appeared in the reaction system, which is proportional to the L-Cysteine concentration. The reaction was monitored spectrophotometrically by measuring the change in absorbance of MCP at 525 nm. The chemical and FIA variables were studied and optimized using the univariate and Factorial design optimization methods. Under the optimum conditions, L-Cysteine can be determined in the range of 0.0500-13.0 µg mL-1. The limit of detection (3ϭ) for L-Cysteine was 0.032 µg mL-1. The relative standard deviation for six replicate determination of 0.500, 5.00 and 12.0 µg mL-1 L-Cysteine were 2.04%, 0.66% and 0.33%, respectively. The proposed method was applied to determinate of L-Cysteine in water, humen serum and urine sample with satisfactory results.