Over the past decade, fibroblast activation protein (FAP), a type II transmembrane serine hydrolase, has been identified and investigated widely in multiple research fields, including biochemistry, pharmacy, clinical medicine, which plays a key role in the metabolism of multiple endogenous peptides and peptide drugs. With the researching of FAP on its physiological functions and the relationships between FAP and clinical diseases, the biological or pathological processes of FAP in normal human or FAP-related diseases has been more clearer. Besides of being a potential therapeutic target for the treatment of cancer, FAP also serves as diagnostic markers for some diseases such as tumor and rheumatoid arthritis. Therefore, a variety of FAP detection methods and inhibitors have been reported. Based on FAP structural features, catalytic properties, endogenous substrate characteristics and exogenous substrate preferences, tissue distribution and specificity, and biological functions, this review not only analyzed the advantages and disadvantages of current FAP detection methods (in vitro and in vivo) and inhibitors but also summarized the structural characteristics of FAP detection substrates and the potential structure-activity relationship of inhibitors. This review will provide an overview of reference values and important reference value for the development of FAP-specific detection methods and potent inhibitors.