A new dispersive liquid-liquid microextraction procedure combined with UV-visible spectrophotometry is described for sensitive and selective determination of rhodamine B. Various analytical parameters such as pH, extraction and dispersive solvents type and volume, sample volumes, and extraction time were optimized. Matrix effects of concomitants were also examined. Preconcentration factor was found to be 330. Detection limit was calculated as 2.1 mu g L-1. The relative standard deviation was found to be 4%. The validation of the developed procedure was checked by the addition/recovery tests. The procedure was successfully performed to the determination of trace levels of rhodamine B in drug, ink, food, cosmetic product, and waste waters.