Abstract: Fructosyltransferase (FTase) production was maximized by Bacillus subtilis NCIM 2439 in submerged fermentation using molasses, which is the renewable carbon substrate. Response Surface Methodology (RSM) involving Central Composite Design (CCD) was adopted to evaluate the activity of fructosyltransferase, by most important factors, such as molasses, glucose and (NH₄)₂SO₄. The optimal set of conditions for maximum fructosyltransferase production was as follows: molasses 23.39 (%v/v), glucose 5.23 g L^-1 and (NH₄)₂SO₄ 1.52 g L^-1. A maximum Fructosyltransferase activity of 78.92 U mL^-1 was obtained at these optimal conditions.