This study was conducted to investigate the extraction of metallothionein (MT) from oyster (Crassostrea plicatul) and optimize its separation and preparation process. According to the single factor experiment and the Box-Behnken experimental principle, the response surface methodology was performed in this experiment, with the material ratio of the oyster tissue to extract, the extraction temperature, the pH value and buffer concentration were taken as the experimental factors, and the extraction capacity of MT was taken as the response value to perform optimization analysis for the extraction process of metallothionein from oyster (Crassostrea plicatul) based on response surface methodology. The results showed that the effects of extraction conditions on the amount of metallothionein extraction descending order were as follows: the ratio of oyster tissue to extraction buffer > pH > extraction temperature = buffer concentration, and then the optimum extraction process parameters were as follows: the material ratio of was 1:4, the value of pH was 9.0, the extraction temperature was 30℃, the concentration of buffer was 0.25 mol/L, and the extraction time was 2 h. Under these conditions, the obtained MT was 0.221 mg/g, which were almost consistent with the predicted data of the model. These results could provide a reference and theoretical basis for the separation and extration of marine metallothionein in addition to its further exploitation and application of the activity.