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Annatto color is one of the oldest of natural carotenoids that have wide applications in food technology. Annatto color is the second natural color additives used in world industry, of economic. It is predicted that demand for it is increasing day by day. Color extraction from annatto seed is done by conventional methods, mainly. Little information is available about the use of new techniques such as response surface methodology of ultrasound technology in the extraction of annatto color. The subject of this study was to optimize the ultrasound-assistant extraction process of annatto color and determination of the best process conditions by using response surface methodology and central square design. The central composite design was used in order to evaluate the effect of extraction temperature (80-20 °C), extraction time (10-2 min), seed to solvent ratio (5-20 %) and duty cycle (0.2-0.8 s), on efficiency of dye extraction from annatto seed and to optimize the extraction process. ANOVA shows the seed to solvent ratio variable (X4) and it square effect (X4 2) have the greatest impact on dye extraction from the annatto seeds. In temperature of 72.7 °C, extraction time of 7.25 min, the seed to solvent ratio of 14 percent and duty cycle of 0.8 s were found as the optimum conditions of the ultrasound-assistant extraction process in order to obtain the maximum efficiency of color under which the absorbance value and extraction efficiency was determined as 0.865 and 6.35 percent of annatto seed, respectively. [ABSTRACT FROM AUTHOR]