Citric acid production by Aspergillus niger NRRL 567 from whey-based medium with different concentrations of supplementing nutrients was studied. The effect of nutrients including glucose, (NH₄)₂SO₄ and KH₂PO₄ on citric acid production was evaluated using statistically-based optimization method and found that glucose and KH₂PO₄ supplementation significantly influenced citric acid production, while (NH₄)₂SO₄ had insignificant effects. The statistical analysis showed that the optimum medium containing 60 g/L of glucose, 1.5 g/L of (NH₄)₂SO₄ and 8.1 g/L of KH₂PO₄ predicted the maximum citric acid production of 12.0 g/L at 240 hr. The predicted citric acid production matched well that measured citric acid production (12.4 g/L) of the validation experiment. The results showed the effectiveness of the statistically-based optimization method and the optimized level of nutrients increased citric acid production by 2 folds, compare with the basal whey medium with 60 g/L glucose.