The isolation of microplastics from other organic and inorganic contents is a major hurdle to the effective quantification and characterization of microplastics in wastewater and biosolids samples. As a result, a well-established and standardized isolation method is vital for the analysis of microplastics. In this study, we evaluated biological hydrolysis, enzymatic hydrolysis, wet peroxidation and ethylenediaminetetraacetic acid treatment for microplastics isolation and demonstrated that the integration of these processes can effectively remove organic and inorganic contents to provide a clear microscope view for microplastics identification from wastewater and sludge samples. To the best of our knowledge, this study is the first to introduce biological hydrolysis and ethylenediaminetetraacetic acid treatment for the isolation of microplastics from environmental samples. The results reported could facilitate the establishment of a standardized procedure for microplastic isolation from wastewater and biosolid samples.