Biofuels are renewable and environmentally friendly, but high production cost makes them economically not competitive, and the development of robust strains is thus one of the prerequisites. In this article, strain improvement studies based on the information from systems biology studies are reviewed, with a focus on their applications on stress tolerance improvement. Furthermore, the contribution of systems biology, synthetic biology and metabolic engineering in strain development for biofuel production is discussed, with an expectation for developing more robust strains for biofuel production.