The release of 1,4-dioxane probably contributes to the deterioration of marine habitats, animal health, and human liver and kidneys. The formulation of 1,4-dioxane in glycols, which has been applied for dehumidifying agents in refineries, may need to be replaced to ensure public health. Further, it is necessary to identify and precisely determine the levels of 1,4-dioxane in glycols for food quality control and environmental safety regulation. The objectives of this study were to validate a liquid–liquid extraction (LLE) method for 1,4-dioxane analysis and to optimize the LLE conditions using a response surface methodology (RSM). With consideration of the food matrix and its applications, polyethylene glycol 200 was used as the model system and analyzed by gas chromatography with flame ionization detection. In the experiments for the optimum extraction temperature and time of 1,4-dioxane in ultrasonic treatment, they were 20 °C and 10 min, respectively. The experimental conditions and results were analyzed by RSM with the Box–Behnken design, and the optimal extraction conditions for the LLE were determined to be coded with three independent variables (sample weight, solvent volume, and centrifugation speed). The amount of 1,4-dioxane increased as the amount of sample increased, whereas the amount of 1,4-dioxane decreased as the amount of solvent increased. This information can help to find the analytical methods for regulating the 1,4-dioxane content and its precise quantification in food products.