Abstract: This study used Sepiella japonica as the research object to establish and optimize the acquisition method of high-turbidity water DNA (environmental DNA, eDNA) in Zhoushan offshore by absolute quantitative technique. The results indicated that the eDNA yield by ethanol precipitation method is 1.76—2.53 times higher than that of filtrating method, but the limitations of the collection volume, treatment requirement and supporting equipment make the ethanol precipitation method difficult to employ in practice. Filter screen with small aperture have no filtering effect on sediment. The size of filter aperture has a great effect on eDNA yield only when small volume water samples were collected. Precipitation treatment to water sample enhanced the yield of eDNA, but it also increased the variation of eDNA yield. Cationic surfactant significantly inhibited eDNA degradation. The effect of the membrane removal method is better than the membrane method, and it is recommended to increase the centrifugation time when the membrane removal method was used. Although the phenol extraction method can not improve the eDNA yield, it can significantly improve the purity of the product. This study is the first to establish an optimal method for obtaining eDNA of macro-organisms from Zhoushan offshore water, which provides reference for water sample collection and eDNA extraction in similar waters.