Abstract: The production of β-glucosidase by Aspergillus niger FTCC 5003 from palm kernel cake as a substrate was studied in solid substrate fermentation using a laboratory column bioreactor. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and airflow rate on the production of β-glucosidase were evaluated using response surface methodology on the basis of a central composite face-centered design. A total of 18 experiments were carried out in which Aspergillus niger FTCC 5003 was cultivated on PKC in an aerated column bioreactor for 7 days under incubation temperature, moisture level and aeration rate determined. Experimental results showed that the highest activity of β-glucosidase (52.06 U g-1) was obtained at an incubation temperature, 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-glucosidase (p<0.01). The statistical results also showed that the interaction effect between initial moisture level and aeration rate significantly influenced the production of β-glucosidase (p<0.05). Optimum conditions suggested by the second-order polynomial regression model for attaining maximum β-glucosidase production were 31.6°C incubation temperature, 57.0% initial moisture and 0.5 L min-1 aeration rate with a predicted production value of 47.20 U g-1.