Biological monitoring of marine microplastic pollution based on mussel indicator is a monitoring method with broad application prospects. However, the current pretreatment process includes a variety of mussel tissue digestion and microplastic density separation technologies, and the scientific nature of pretreatment technology has yet to be verified, which makes it difficult to guarantee the accuracy of marine microplastic pollution monitoring results obtained by this method and the data are difficult to compare. In order to comprehensively evaluate the accuracy of multiple mussel tissue digestion and microplastic density separation operations and to obtain a cost-effective and reliable pretreatment technology, comparison tests of 4 common digestion methods (mixed acid digestion, potassium hydroxide digestion, Fenton digestion and protease K digestion) and density separation tests of 3 microplastic flotation fluids (sodium chloride saturated solution, sodium iodide saturated solution and potassium formate saturated solution) were carried out. The effects of different digestion methods on the digestion efficiency of mussel tissue and on the morphology, spectral characteristics and recovery rate of common microplastic in the ocean, as well as the separation effect of microplastic in different flotation fluids were evaluated, and the digestion conditions were optimized by single factor and response surface tests. The results showed that the Fenton digestion method had both efficient digestion of mussel tissue and low destructive effect on microplastic, and could be used as the optimal method for digestion of mussel tissue. After optimization, under the conditions of H₂O₂ (30%) volume 40 mL, Fe²⁺ concentration 0.020 mol/L, temperature 59℃, pH 3.7 and digestion time 24 h, the digestion rate of 10 g mussel tissue reached 96.7%. At the same time, this study confirmed that potassium formate saturated solution could replace sodium chloride and sodium iodide saturated solution as the flotation fluid with high efficiency. The development of the above research provides a reference for the improvement and standardization of the pretreatment technology for biological monitoring of marine microplastic pollution based on mussel indicator.