A new cloud point extraction procedure was developed as surfactant using mixed micelles of Tween-80 and Tergitol TMN-6 for separation and preconcentration of crystal violet in aqueous media. To determine the optimum conditions, the effects of analytical parameters such as pH, mixed ratio of surfactants, amount of mixed surfactants, the equilibrium temperature and time, centrifugation time and rates, and matrix effect of some ions were studied. Linear calibration curve (range from 2 to 400 ng ml(-1)) was obtained after applying developed cloud point extraction procedure. Crystal violet content of some waste water and lake water samples was determined after using developed method by UV-vis. spectrometry at 600 nm.