Levansucrase, enzyme produced by Bacillus subtilis, synthesizes levan using sucrose as substrate. Levan is formed by transfructosylaction reaction of levansucrase, units of fructosyl are connecting linked through β-(2→6) bonds in linear chain and β-(2→1) bonds in ramifications. Potential applications of levan have been proposed in the cosmetics, foods and pharmaceuticals fields. Response surface methodology, a statistical tool, has been an alternative to the empirical study for enzyme and polysaccharides production optimization. The present study aim was to assess the effects of the sucrose and Na₂SO₄ concentrations and pH on levansucrase production, using statistical methodology. Central composite design 2³ including 6 axial points and 2 repetitions at the central point statistical model was applied to optimize levansucrase production, varying fermentation conditions of sucrose concentration (25 - 395 g.L^-1), pH (4.32 - 7.68) and Na₂SO₄ concentration (0 – 0.4 M). Fermentations were carried out in batch culture medium containing (g.L^-1): yeast extract 2; KH₂PO₄ 1; (NH₄)₂SO₄ 3; MgSO₄ (7H₂O) 0.6; MnSO₄ 0.2; citrate ammonium, in 150 mL Erlenmeyer flasks containing 50 mL culture medium. Cultures were harvest by centrifugation and supernatant was defined as cell-free extract (CFE) and was used to determine levansucrase. Response variable studied, levansucrase activity, were analyzed using STATISTICA 7.0 software at the 5% significance level. Levansucrase activity was estimating by levan-forming. The reaction mixture: CFE (250 μL), sucrose 1 M (250 μL) and acetate buffer 50 mM, pH 5 (500 μL) at 30°C for 2 h. One unit of enzyme activity (AU) is expressed as the amount of enzyme that polymerizes 1 μmol of reducing sugar per 1 min, under experimental conditions experimental conditions. Levan was estimated. The statistical model showed R²=0.975. Levansucrase activities ranged from 0,89 to 11,89 AU in sucrose medium and the highest activities observed occurred in sucrose 395 g.L^-1, pH 6.0, Na₂SO₄ 0,25 M. The sucrose was the most important factor on levansucrase production by Bacillus subtilis natto.