Increasingly serious energy and environmental problems have led to a growing interest in the study of biomass energy. Isobutanol (2-methyl-1-propanol) has become a new generation of biofuel with excellent characteristics such as high octane number, high energy density, low vapor pressure, low hygroscopicity, etc., and it is an ideal component for gasoline blending. However, this important compound is mainly produced by chemical synthesis, which not only consumes limited non-renewable human resources but also pollutes the environment. Compared with traditional chemical synthesis, microbial fermentation has the advantages of mild conditions, easy operation, few by-products, environmental protection, energy saving, and cost reduction, especially the easy availability of raw materials and the utilization of renewable resources indicating its promising development prospect. This review summarized the production of isobutanol by fermentation, especially the pioneer work on improving the isobutanol titer in the fermentation broth. For the bioisobutanol fermentation, another challenge is to recover this compound from the fermentation broth efficiently. The current methods using distillation have the disadvantage of being too energy-intensive, raising the cost of the fermentation method. Adsorption, gas stripping, membrane osmotic vaporization, membrane extraction, and liquid-liquid extraction, including salting-out, are new methods for the separation of bio-based isobutanol, which are very important for more efficient recovery of bio-isobutanol. The advantages and disadvantages of each separation technique were summarized. The large amount of water in the isobutanol fermentation broth results in high energy consumption for its separation, and also reduces the sugar load of the fermentation, and increases the amount of water used for fermentation. Further development of microorganisms that can increase solvent titer or methods for in-situ product removal should be developed to improve the market competitiveness of bioisobutanol..