Syntrophic metabolism is one of the important interspecies relationships among microbes. Syntrophic microorganisms not only distribute in soils, freshwater, marine anoxic sediments, anaerobic digestion and gastrointestinal tract of animals, but also present in extreme environments such as subsurface oil reservoirs. They play essential roles in anaerobic degradation of organic compounds to methane and carbon dioxide. Study on the syntrophic metabolisms of syntrophic microorganisms through culture-dependent methods, would help understand the biogeochemical cycle of elements in anoxic environments, and deal with the global energy crisis and global warming problems. However, it is difficult to isolate syntrophic microorganisms for their slow-growing and oxygen-sensitive properties. This review summarizes the recent studies on the isolation strategies of syntrophic microorganisms, and their physiological and biochemical properties. Furthermore, the future development trend of culture techniques including high throughput screening and targeted isolation of syntrophic microorganisms were discussed.