Kluyveromyces lactis is a representative species of unconventional yeast, and has very important applications in basic and applied microbiology. It can also serve as a promising platform for various industrial applications owing to its advantages such as its high level of food safety, powerful capability for protein secretion, stable high-level expression of heterologous genes and excellent fermentation characteristics on a large scale. At present, K. lactis has been widely used as a host for protein expression in the food and pharmaceutical industries. In recent years, using this yeast as a biotechnological chassis, biotechnological researchers have successfully constructed efficient microbial cell factories capable of producing various biochemicals by using synthetic biology techniques. This technology shows great potential for a wide range of applications. In this review, we describe the metabolic characteristics, synthetic biology elements, genetic manipulation tools and gene editing strategies of K. lactis. Furthermore, the application of K. lactis as a microbial cell factory for the production of high value-added products is reviewed. It is believed that the information provided in this article will make a significant contribution to the advancement of the high-efficiency production of industrially important biochemicals by the engineered cell factories of K. lactis.