The ultimate goal in the production of biofuels is to produce fuels identical or similar to petroleum-derived transportation fuels more efficiently and in commercial quantities. Synthetic biologists have been engineering microbes to synthesize biofuels, such as butanol and fatty acid- or isoprenoid-based fuels, which are nearly identical to gasoline and diesel. One of the most urgent demands along this direction is to attain a solid framework for characterizing and standardizing the biological parts and devices. It seems quite promising because biotechnologies specially based on miniaturizations have been making a big contribution to this work. Therefore, in this review, recent advances and difficulties in the biofuel field are discussed, along with the advances of synthetic biology, which will make it possible to create designer microorganisms that produce economically viable next generation biofuels, aside from bioethanol, from corn or sugar cane, and biodiesel from plant or animal oils.