Abstract: Aspergillus niger FTCC 5003 was cultivated on palm kernel cake as a substrate to produce β-xylosidase in solid substrate fermentation. The concurrent effects of three independent variables, including incubation temperature, initial moisture content of substrate and airflow rate on the production of β-xylosidase were studied by response surface methodology. A number of 18 trials were carried out in which solid cultivation of substrate was performed in an aerated packed-bed bioreactor for 7 days. Experimental results showed that the highest activity of β-xylosidase (6.13 U g-1) was obtained at an incubation, an initial moisture level and an aeration rate of 32.5°C, 60% and 1.5 L min-1, respectively. Statistical analysis revealed that the quadratic terms of incubation temperature and initial moisture content had highly significant effects on the production of β-xylosidase (p<0.01). Optimum conditions suggested by the statistical model for the production of β-xylosidase were 32.3°C incubation temperature, 59.7% initial moisture and 0.5 L min-1 aeration rate for attaining a predicted production level of 5.32 U g-1.