Describing predator-prey relationships among organisms is essential in the food web studies of aquatic ecosystems. The traditional method of food web research is based on the morphological observation of the stomach contents. With the development of science and technology, new methodological approaches such as stable isotope analysis, fatty acid profiling, and DNA barcoding effectively avoid the limitations of stomach content analysis, provide a deeper understanding of the feeding characteristics of aquatic organisms, and have been widely used in feeding ecology studies. In this paper, the development trend of the research methods used to study aquatic food webs was introduced. The current status of stable isotope analysis, fatty acid profiling, and DNA barcoding techniques, combined with stomach content techniques, were reviewed, focusing on the application and development of the four methods, the comparison of their advantages and limitations, their applicable scope and assumptions, and the requirements for available experimental samples. These areas are discussed in terms of the assumptions made when applying these techniques to the study of aquatic ecology and the combinations of these techniques that investigators use for different study objectives. Recommendations are made for future aquatic feeding ecology experimental work that would improve understanding of food web structures and the functioning of aquatic ecosystems. As the traditional research method, stomach content analysis remains an irreplaceable technique in the study of feeding ecology. Stable isotopes, fatty acid profiling, DNA barcoding, and other molecular techniques are strong complements to stomach content analysis and provide additional useful information for reconstructing aquatic food webs, which would be helpful in ecosystem-based fishery management.