The gastrointestinal tract is the largest hormone endocrine organ in the body, and gastrointestinal hormones are widely involved in regulating physiological metabolism, disease progression, and the occurrence and development of tumors. In order to accurately measure gastrointestinal hormones, this review briefly describes the physiological characteristics of gastrointestinal hormones. Secondly, the principles of different measurement methods are discussed, including bioassay, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), mass spectrometry (LC-MS/MS), and process independent analysis (PIA). Finally, the research on the conversion of gastrointestinal hormones and the diagnostic value of gastrointestinal hormone measurement are discussed. The conclusion drawn from the review is that measuring gastrointestinal hormones is not only closely related to checking digestive function and diseases, but also important for monitoring extraintestinal function. In addition, although MS detection technology has good application prospects, it is still limited by expensive instrument costs and operational complexity. Therefore, current immunoassay technologies (RIA and ELISA) are still the most feasible methods for measuring gastrointestinal hormones in biological fluids