The industrial microorganisms used for fructooligosaccharides (FOS) synthesis are generally fermented with sucrose as carbon source to induce the production of β-fructofuranosidase (FFase) having transfructosylation activity. Consequently, isolation of novel FFase producers from a sucrose-enriched biotope would help improve FOS productivity and reduce the process cost. Here, three fungi isolated from a unique sugarcane molasses habitat were found to possess FFase activity and one of them, XG21, exhibited a high capacity to synthesize FOS. Analysis of its morphological properties and ribosomal internal transcribed spacer (ITS) sequence allowed the taxonomic position to be assigned and it was thus identified as Aspergillus tubingensis XG21. It could utilize various potential carbon sources for vigorous growth, but only produced high-level FFase activity on sucrose. Furthermore, the transfructosylation ability and FOS synthesis were analyzed by TLC and HPLC. During the transfructosylation reaction, an increase in sucrose concentration led to the remarkable enhancement in FOS formation with the maximum content of up to 56.9% within 8 h. Finally, the sugarcane molasses was used to cultivate A. tubingensis XG21 and the optimal FFase activity reached up to 558.3 U/g, which was 88.9% higher than that with sucrose as carbon source. These results indicate that A. tubingensis XG21 can be considered as a new genetic resource adapted to cheaply available carbon sources for FOS production.