Among fifteen different nutritional factors, results of statistical analysis that obtained by Plackett– Burman design showed that sucrose, ZnSO4 and MnSO4 were the most promising factors affecting levansucrase production by Bacillus subtilis M with confidence levels more than 97%. For optimum concentrations of the selected variables, central composite design (CCD) was applied. Variance analysis (ANOVA) showed a determination coefficient (R2) value of 0.86 for levansucrase and 0.911 for levan production ensuring a satisfactory adjustment of the quadratic model with the experimental data. A high similarity was observed between the predicted and experimental results reflected the accuracy and model applicability for optimization. The correlation analysis showed that there was 83% correlation between levansucrase production and levan production yield. In compare to the basal medium, overall 7.58 and 7.82 folds increase in levansucrase and levan respectively. Investigation of in vitro levan antioxidant activity using different methods showed that 56.25 ± 0.57 % DPPH inhibition was achieved at 4.5 mg/ml concentration. Concentration 2.5 mg/ml exhibited strongest reducing power (1.023± 0.015) of Fe3+ (CN-)6. Highest FRAP value (2669 ± 74.63 μmol TE/ 100 g) was obtained at (2.5 mg /ml) levan. Free radical scavenging activity of levan against ABTS+ cation radical was recorded.