β(1→3)-d-Glucan sensors were fabricated using bi-enzyme and tri-enzyme immobilized systems with gold nanoparticles (GNPs) to increase sensitivity. The plant β(1→3)-d-glucanase (βG), glucose oxidase (GOD) or/and peroxidase (POD) in agarose–corn flour–gelatin (ACG) matrix were coated on platinum disc electrode to detect soluble β(1→3)-d-glucan. The atomic force microscopy (AFM) revealed that GNPs embedded in ACG formed tiny islands/clusters with enzymes. Both of bi-enzyme sensor (ACG–βG–GOD–GNPs/Pt) and tri-enzyme sensor (ACG–βG–GOD–POD–GNPs/Pt) had response time less than 20s for β(1→3)-d-glucan. A linear calibration plot for bi-enzyme sensor was obtained for β(1→3)-d-glucan concentration ranged from 100 to 1000ngmL⁻¹ (R²=0.983). The lower detection limit was 30ngmL⁻¹ using applied potential of 200mV and scan rate of 50mVs⁻¹; with signal to noise ratio (S/N) of 3. Fabricated tri-enzyme sensor was also operable under similar conditions with LOD of 50ngmL⁻¹ (R²=0.989) at −175mV applied potential and scan rate of 50mVs⁻¹. Both sensors were durable and could be repeatedly used for at least 14 times. When the tri-enzyme sensor was employed to analyze β(1→3)-d-glucan content in alcoholic beverages, the results were comparable to those obtained by standard method.