Aspergillus niger ATCC20611 is one of the most potent filamentous fungi used commercially for production of fructooligosaccharides (FOS), which are prospective components of functional food by stimulating probiotic bacteria in the human gut. However, current strategies for improving FOS yield still rely on production process development. The genetic engineering approach hasn't been applied in industrial strains to increase FOS production level. Here, an optimized polyethylene glycol (PEG)-mediated protoplast transformation system was established in A. niger ATCC 20611 and used for further strain improvement. The pyrithiamine resistance gene (ptrA) was selected as a dominant marker and protoplasts were prepared with high concentration (up to 10g wet weight mycelium) by using mixed cell wall-lysing enzymes. The transformation frequency with ptrA can reach 30-50 transformants per μg of DNA. In addition, the efficiency of co-transformation with the EGFP reporter gene (egfp) was high (approx. 82%). Furthermore, an activity-improved variant of β-fructofuranosidase, FopA(A178P), was successfully overexpressed in A. niger ATCC 20611 by using the transformation system. The transformant, CM6, exhibited a 58% increase in specific β-fructofuranosidase activity (up to 507U/g), compared to the parental strain (320U/g), and effectively reduced the time needed for completion of FOS synthesis. These results illustrate the feasibility of strain improvement through genetic engineering for further enhancement of FOS production level.