The relationship between biological materials and the immobilization matrix is a critical point in attaching biomolecules onto transducers in the preparation of biosensors. Various types of chemical molecules are used to conjugate biological compounds in the design of biofunctional surfaces. Among them, chemobrionics (CBCs) are good alternatives as an immobilization matrix to improve performance parameters of biosensors such as linear range, limit of detection (LOD), stability, reproducibility, etc. Here, CBCs are used to develop enzymatic biosensors, and glucose oxidase (GOx) is selected as a model enzyme. Firstly, CBC is functionalized using APTES to obtain free amine groups for immobilization of GOx via covalent bonds. The synthesized CBC-APTES is used as a matrix for the conjugation of GOx using glutaraldehyde as a crosslinker. The proposed CBC-APTES/GOx is formed on the glassy carbon electrode, and its performance is checked for glucose detection. The linear range is 0.025–1.25 mM with an LOD of 0.024 mM Glc. After finding no effect of some potential interference on the current response, CBC-APTES/GOx is successfully applied to analyze Glc in artificial samples such as serum, urine, sweat, and saliva. The objective here is to outline the integration of CBC in biosensor preparation, which is successfully carried out for the first time in literature.