Exacerbation of climate change and air pollution around the world have emphasized the necessity of replacing fossil fuels with clean and sustainable energy. Metabolic engineering has provided strategies to engineer diverse organisms for the production of biofuels from renewable carbon sources. Although some of the processes are commercialized, there has been continued effort to produce advanced biofuels with higher efficiencies. In this article, metabolic engineering strategies recently exploited to enhance biofuel production and facilitate utilization of non-edible low-value carbon sources are reviewed. The strategies include engineering enzymes, exploiting new pathways, and systematically optimizing metabolism and fermentation processes, among others. In addition, metabolic and bioprocess engineering strategies to achieve competitiveness of current biofuel production systems compared with fossil fuels are discussed.