In this paper, a central composite design was applied to optimize the bioleaching of iron from a kaolin sample containing 2.2% iron impurity by Aspergillus niger isolated from pistachio shell. The strains were inoculated into 500 ml flasks containing 100 ml media consisted of (g/l): sucrose 120; NH4NO3 0.45; KH2PO4 0.1; MgSO4.7H2O 0.3; FeSO4.7H2O 10-4; ZnSO4.7H2O 25×10- 5. The effects of initial pH, sugar and spore concentrations on iron removal extent were investigated. The two-level factorial design points were pH 2 and 5, sugar conc. 70 g/l and 130 g/l, spore conc. 9×107 and 35×107 spores/l. Also, the increase of dissolved iron, oxalic acid concentration, changes in pH value, and sugar concentration were registered. Consequently, after 10 days, the iron concentration of the best condition reached to 179.3 ppm that means 38.8% of the total iron content is removed. Furthermore, the data analysis showed that all the factors are significant, and the iron removal extent increases by increasing the initial pH to 4.4, sucrose content to 93.8 g/l, and spore concentration to 305.5 spores/μl, but further increase in each factor value has negative effect on the response.