2,3-Butanediol has been known as a platform green chemical, and the production cost is the key problem for its large-scale production in which the carbon source occupies a major part. Sugarcane molasses is a by-product of sugar industry and considered as a cheap carbon source for biorefinery. In this paper, the fermentation of 2,3-butanediol with sugarcane molasses was studied by reducing the medium ingredients and operation steps. The fermentation medium was optimized by response surface methodology, and 2,3-butanediol production was explored under the deficiency of sterilization, molasses acidification, and organic nitrogen source. Based on these experiments, the fermentation medium with sugarcane molasses as carbon source was simplified to five ingredients, and the steps of molasses acidification and medium sterilization were reduced; thus, the cost was reduced and the production of 2,3-butanediol was enhanced. Under fed-batch fermentation, 99.5 g/L of 2,3-butanediol and acetoin was obtained at 60 h with a yield of 0.39 g/g sugar.