Abstract: In this paper, a new viewpoint on the activity determination of β-galactosidase is reported. Glucose oxidase was directly immobilized on a glassy carbon electrode and mediated by ferrocene. The biosensor's performance was based on mediated electron transfer by ferrocene, which reduced via glucose oxidase reaction. In this reaction, substrate of glucose oxidase, glucose was provided by the activity of β-galactosidase in the sample. The parameters of the fabrication process for the electrode were optimized. Experimental conditions influencing the biosensor performance, such as pH, ferrocene and lactose concentrations, and temperature, were investigated and assessed. Finally, the biosensor was successfully applied to determination of β-galactosidase activity of artificial intestinal juice.