Isothiocyanates are a class of secondary metabolites mainly derived from cruciferous plants. They give food a unique flavor and exhibit a variety of biological activities, such as anticancer, antidiabetic, neuroprotective, antimicrobial, anti-inflammatory, and antioxidant properties. They are widely used in food, medicine, and agriculture. The yields of isothiocyanates from natural sources are generally low, while chemical synthesis often leads to significantly contaminated isothiocyanates. In the meantime, with the development of synthetic biology, new methods have emerged for the green, efficient, and stable production of isothiocyanates. In this review, the classification, biological activities, bioavailability, biosynthesis pathways, and molecular biology methods that improve the yields of isothiocyanates in species are systematically expounded in detail. The aim is to deepen the understanding of the role and value of isothiocyanates and to provide technical support for their further development and utilization. This study is expected to provide a theoretical basis for the development of functional foods as well as insights into the high-yield preparation of isothiocyanates.