This study investigated the effect of low-cost carbon and nitrogen sources on fructosyltransferase (FTase) production by Aspergillus oryzae IPT-301, aiming to optimize the enzymatic synthesis of fructooligosaccharides (FOS), prebiotic compounds valued for their bifidogenic effects. FTase is a key enzyme in transfructosylation, the central step in FOS production. To reduce production costs, Very High Polarization (VHP) and Demerara (DM) sugars were evaluated as carbon sources, while sodium nitrate (NaNO₃), ammonium sulfate (NH₄)₂SO₄, and urea were tested as nitrogen sources. FTase production, both extracellular and intracellular, was conducted under submerged fermentation at 30 °C and 200 rpm for 72 h. DM sugar outperformed VHP, increasing extracellular and intracellular transfructosylation activity (A_T) by 2.3-fold and 2.1-fold, respectively. Among nitrogen sources, NaNO₃ was most effective in DM-containing media, yielding 1.6–2.0 times higher extracellular A_T and up to 4.7 times greater intracellular activity compared to other nitrogen sources. These findings suggest that the combination of DM sugar and NaNO₃ significantly enhances FTase yield, providing a cost-effective strategy for industrial-scale FOS production.