Microplastics pollution in aquatic ecosystems has aroused increasing global concern, leading to an explosive growth of studies regarding microplastics published in the past few years. To date, there is still a lack of standardized methodologies used for the detection of microplastics within environmental samples, thus hampering comparison of the reported data. This review summarizes the currently used methodologies for sampling, extracting and identifying microplastics in three kinds of aquatic environmental matrices (water, sediment and aquatic biota) and includes a critical discussion of the advantages and limitations of these methodologies. The quality control and quality assurance measures taken to reduce background contamination and validate analytical methods are also discussed. Finally, this review highlights the current challenges and gives suggestions for the future research.