The global energy crisis and the urgent need to mitigate climate change have intensified the search for sustainable alternatives to fossil fuels. Biofuels, derived from biological sources, present a promising renewable solution. However, first-generation biofuels from food crops face challenges related to land-use change, water consumption, and the “food-vs-fuel” debate. Second-generation biofuels, using non-food lignocellulosic biomass, offer a more sustainable pathway but are limited by recalcitrant biomass and inefficient conversion. Synthetic biology, the engineering of biological systems for novel functions, is transforming the biofuel landscape by providing tools to overcome these barriers. This review explores the latest advances in synthetic biology for sustainable advanced biofuel production. We detail core engineering principles, including pathway design, modular cloning, and CRISPR-based genome editing, that enable precise rewiring of microbial metabolism. We focus on engineering various host chassis—from traditional workhorses like   Escherichia coli   and   Saccharomyces cerevisiae   to non-model organisms like   Clostridium   and photosynthetic cyanobacteria—for producing diverse fuel molecules beyond ethanol, such as advanced alcohols, fatty acid-derived alkanes/alkenes, isoprenoid-based fuels, and hydrogen. We also discuss the integration of consolidated bioprocessing (CBP) and the use of one-carbon (C1) feedstocks like CO₂ and methane to enhance sustainability and reduce costs. Finally, we address persistent challenges in yield, titer, productivity, and scale-up, and outline future perspectives on integrating systems biology, machine learning, and circular bioeconomy principles to design the next generation of biofuel production systems.