An object of the present invention is to provide a more practical method for extracting substances from a fecal sample. More specifically, an object of the present invention is to provide a method for extracting substances from a fecal sample, the method being suitable for conducting analyses such as metabolomic analysis using mass spectrometry or nuclear magnetic resonance spectrometry, and metagenomic analysis using a next-generation sequencer. The present invention provides a method for extracting one or more substances from a fecal sample, comprising the following steps A to C: step A: a step of suspending the fecal sample in any one of the following solvents (a) to (g) to obtain a suspension: (a) a solvent comprising an aqueous solvent and a hydrophilic organic solvent； (b) a solvent comprising an aqueous solvent and a hydrophobic organic solvent； (c) a solvent comprising an aqueous solvent, a hydrophilic organic solvent and a hydrophobic organic solvent； (d) a solvent comprising a hydrophilic organic solvent； (e) a solvent comprising a hydrophobic organic solvent； (f) a solvent comprising a hydrophilic organic solvent and a hydrophobic organic solvent； and (g) a solvent consisting of water； step B: a step of separating a liquid layer comprising the solvent from the suspension； and step C: a step of removing proteins from the liquid layer and then obtaining the one or more substances.
