The lipase produced by the Aspergillus niger strain AC-54 has been widely studied due to its enantioselectivity for racemic mixtures. This study aimed to optimize the production of this enzyme using statistical methodology. Initially a Plackett-Burman (PB) design was used to evaluate the effects of the culture medium components and the culture conditions. Twelve factors were screened: water content, glucose, yeast extract, peptone, olive oil, temperature, NaH2PO4, KH2PO4, MgSO4․7H2O, CaCl2, NaCl, and MnSO4. The screening showed that the significant factors were water content, glucose, yeast extract, peptone, NaH2PO4, and KH2PO4, which were optimized using response surface methodology (RSM) and a mathematical model obtained to explain the behavioral process. The best lipase activity was attained using the following conditions: water content (20%), glucose (4.8%), yeast extract (4.0%), and NaH2PO4 (4.0%). The predicted lipase activity was 33.03 U/ml and the experimental data confirmed the validity of the model. The enzymatic activity was expressed as µmoles of oleic acid released per minute of reaction (µmol/min).