Fructooligosaccharides (FOSs) are excellent food ingredients or feed additives by stimulating probiotics. In this paper, a gene encoding a glucose repressor in the β-fructofuranosidase producer 33 with high-level FOS biosynthesis was disrupted, and glucose repression in disruptant D28 was relieved. The disruptant D28 produced up to 2100 U/mL of β-fructofuranosidase activity, whereas the enzyme activities produced by parent strain 33 and complemented strain C11 were below 600 U/mL. The whole cells of the disruptant D28 was used to convert cane molasses into FOSs, and 0.58 g of FOSs/g of molasses sugar was synthesized from 350 g/L cane molasses sugar within 4 h. Results demonstrated that the industrial waste cane molasses can be efficiently converted into FOSs by the glucose derepression mutant D28 with high β-fructofuranosidase activity. This low-cost and environmentally friendly bioprocess has great potential applications in bioengineering and biotechnology for FOS production.