Polyketides (PKs) and non-ribosomal peptides are the most important drug-leads for human, animal, and plant diseases. The conserved modular architectures and biosynthetic assembly line of polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS) endow PKs and NRPs with extremely diverse structures and activities and bring infinite possibilities to edit and modify the backbone structure of PKs and NRPs by adding, removing, inactivating and replacing PKS/NRPS modules or domains. The biosynthetic machinery of microbial polyketide natural products has evolved delicately with specific recognition and efficient catalysis of upstream intermediates by downstream enzymes/domains. Therefore, manipulations of PKS/NRPS and their related tailoring enzymes usually lead to attenuated production or abolished accumulation of intermediates with modified structures. As the terminal domain of most PKS and NRPS, thioesterases (TEs) play crucial roles in substrate selection during the chain release of these bioactive natural products, serving as pivotal bottleneck steps in their late-stage biosynthesis. TEs mainly perform chain hydrolysis or ester transfer reactions by nucleophilic attack of foreign nucleophiles such as H₂O. Meanwhile, TEs also undergo nucleophilic attack by intramolecular oxygen atom, nitrogen atom, or carbon atom to achieve macrolactonization, macrolactamization, or Claisen condensation, respectively. There are two main classes of TEs involved in natural product biosynthesis. Type I TEs (TEIs) are commonly found in type I cis-AT PKS, trans-AT PKS, NRPS, and fungal PKS/NRPS, which are mainly located at the end module of synthase. In addition to TEIs, there is also a class of free type II TE (TEIIs), which catalyzes the release of incomplete or incorrectly extended intermediates during PKs and NRPs biosynthesis. Besides, a distinct class of free TE was identified in the chain release of polyether backbones, such as monensin and nanchangmycin. Since 2001, more than 20 crystal structures of TEs from diverse PKSs and NRPSs have been solved. The structural elucidation of TEs has unlocked the mystery of their structural and functional interaction, laid the foundation for the TE classification and mechanistic insight into the substrate selectivity and catalytic efficiency of TE, which further promotes the understanding of the chain release mechanism of natural products and better served the rational design of TE. Previous articles have systematically reviewed the structure, function, and regulatory mechanism of different TE families. Horsman et al. also reviewed the diversity, structure, and mechanism of TEs in PKSs and NRPSs. They put forward an insightful view that TEs might act as logic gates for substrate loading and chain releasing during the biosynthesis of natural products. It provides an important perspective for studying the evolution and functional prediction of TEs. This review summarizes the structural characteristics of various TE, focusing on the structural consistency of thioesterase to the catalytic mechanism. Additionally, this review follows the progress and limitations on the catalytic mechanism and computational simulation of type I TE, providing a detailed analysis of the chemical essence of thioesterase-catalyzed chain release reactions. This review aims to deliver revealing suggestions for the structural elucidation and mechanistic insights of TE, as well as its rational design for improved chain release of unnatural products.