Magnetized polysiloxane coated with polyaniline (mPOS–PANI) was used as a support for β-galactosidase immobilization via glutaraldehyde. The galactooligosaccharides (GOS) production by this derivative was investigated under different initial lactose concentrations (5–50%) and temperatures (30–60°C). The initial lactose concentration in the reaction media affected the total amounts of produced GOS and their time course production was described as a “bell-shaped” curve as a result of the balance between transgalactosylation and hydrolysis. No significative difference was observed for the free and immobilized enzymes. The reaction rates for lactose hydrolysis and GOS formation increased with increasing temperature from 30°C to 60°C, but GOS production at all lactose conversion levels was almost unchanged with changing temperature. The mPOS–PANI matrix was also characterized by scanning electronic microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometry (VSM), thermomagnetization, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA).