In the present work, Bacillus subtilis was engineered as the cell factory for isobutanol production due to its high tolerance to isobutanol. Initially, an efficient heterologous Ehrlich pathway controlled by the promoter P-43 was introduced into B. subtilis for the isobutanol biosynthesis. Further, investigation of acetolactate synthase of B. subtilis, ketol-acid reductoisomerase, and dihydroxy-acid dehydratase of Corynebacterium glutamicum responsible for 2-ketoisovalerate precursor biosynthesis showed that acetolactate synthase played an important role in isobutanol biosynthesis. The overexpression of acetolactate synthase led to a 2.8-fold isobutanol production compared with the control. Apart from isobutanol, alcoholic profile analysis also confirmed the existence of 1.21 g/L ethanol, 1.06 g/L 2-phenylethanol, as well as traces of 2-methyl-1-butanol and 3-methyl-1-butanol in the fermentation broth. Under microaerobic condition, the engineered B. subtilis produced up to 2.62 g/L isobutanol in shake-flask fed-batch fermentation, which was 21.3% higher than that in batch fermentation.