Glucose biosensors have become increasingly significant in detecting diabetes in recent years. This paper presents the design and fabrication of an enzymatic, bio-disposable, nontoxic, glucose sensor using a novel Catalytic and Binder Layer (CBL) synthesized with natural material. The Interdigitated Electrodes (IDE) for the proposed glucose biosensor were designed with ‘graphene’ conductive ink screen-printed on a glossy surface of photo-paper. These screen-printed IDEs were further functionalized with Polyaniline (PANI), a conductive polymer material, and a layer of glucose-selective enzymes, i.e., glucose oxidase (GOx). Additionally, a novel CBL was prepared with a composite of green synthesized silver nanoparticles (AgNPs) as a catalytic agent and natural stem fluid of banana flowers as a binder, and it was incorporated onto the screen-printed IDE functionalized with PANI/GOx. The GOx enzymes, when combined with green synthesized AgNPs, facilitate a biphasic catalytic process including two sequential steps. Initially, the GOx enzyme facilitates the conversion of glucose, resulting in the production of hydrogen peroxide (H2O2) as a primary substance. During the second phase, the green synthesized AgNPs catalyze the conversion of hydrogen peroxide (H2O2) into hydroxide ions (OH−), resulting in a localized pH change near the reaction site. The conductivity of PANI is pH-responsive, making it a useful chemiresistive sensor. By detecting the concentration of H2O2, the PANI layer can be used to quantify glucose concentration. The proposed CBL has the advantages of being nontoxic, environmentally friendly, low-cost, and synthesized with available resources.