Abstract: The present disclosure provides a method for detecting short-chain fatty acids in biological samples, including a derivatizing step, a loading step and a detecting step. The derivatizing step includes treating the short-chain fatty acids in the biological sample with 2-nitrophenylhydrazine for derivatizing the short-chain fatty acids into a sample to be detected. The loading step includes loading the sample onto a paper carrier. The detecting step includes analyzing the sample loaded onto the paper carrier by direct analysis in real time mass spectrometry for obtaining a detection result. The method provided by the present disclosure may complete the analysis of the biological sample within a short period of time and achieve a quantitative result comparable to that obtained by conventional chromatographic approaches.