The third-generation (3G) biorefinery aims to use microbial cell factories or enzymatic systems to synthesize value-added chemicals from one-carbon (C1) sources, such as CO 2 , formate, and methanol, fueled by renewable energies like light and electricity. This promising technology represents an important step toward sustainable development, which can help address some of the most pressing environmental challenges faced by modern society. However, to establish processes competitive with the petroleum industry, it is crucial to determine the most viable pathways for C1 utilization and productivity and yield of the target products. In this review, we discuss the progresses that have been made in constructing artificial biological systems for 3G biorefineries in the last 10 years. Specifically, we highlight the representative works on the engineering of artificial autotrophic microorganisms, tandem enzymatic systems, and chemo-bio hybrid systems for C1 utilization. We also prospect the revolutionary impact of these developments on biotechnology. By harnessing the power of 3G biorefinery, scientists are establishing a new frontier that could potentially revolutionize our approach to industrial production and pave the way for a more sustainable future.