A method was developed to determine 2-mercaptobenzimidazole in water and urine samples using dispersive liquid-liquid microextraction technique coupled with ultraviolet-visible spectrophotometry. It was essential to peruse the effect of all parameters that can likely influence the performance of extraction. The influence of parameters, such as dispersive and extraction solvent volume and sample volume, on dispersive liquid-liquid microextraction was studied. The optimization was carried out by the central composite design method. The central composite design optimization method resulted in 1.10 mL dispersive solvent, 138.46 μL extraction solvent, and 4.46 mL sample volume. Under the optimal terms, the calibration curve was linear over the range of 0.003-0.18 and 0.007-0.18 μg/mL in water and urine samples, respectively. The limit of detection and quantification of the proposed approach for 2-mercaptobenzimidazole were 0.013 and 0.044 μg/mL in water samples and 0.016 and 0.052 μg/mL in urine samples, respectively. The method was successfully applied to determination of 2-mercaptobenzimidazole in urine and water samples.