Fructooligosaccharides (FOSs) are excellent food ingredients or feed additives by stimulating probiotics. In this paper, a CREA gene encoding a glucose repressor in the β-fructofuranosidase producer Aureobasidium melanogenum 33 with high-level FOSs 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 environmental friendly bioprocess has great potential applications in bioengineering and biotechnology for FOSs production.