<p>In the present study we explored the synthesis of nano bioconjugates using differently functionalized MWCNTs and glucose oxidase- a model enzyme. The impact of MWCNT functional groups on enzyme kinetics and biosensing property of glucose oxidase were investigated which is crucial for bioelectrode fabrication. Herein, wet chemical methods are used to functionalize pristine multiwalled carbon nanotube (p-MWCNT) by carboxylic group (MWCNT-COOH) and amine group (MWCNT-NH₂). The effect of surface functionalization on covalent immobilization of glucose oxidase (GOx) has been studied using several techniques - SEM, EDAX, FTIR, Raman, UV-visible spectroscopy, Circular Dichroism spectroscopy and electrochemical analysis to characterize the GOx-MWCNT nano-bioconjugates and their biocatalytic properties. The immobilization process was optimized and systematically compared using the Taguchi method - a statistical approach for improving immobilization process performance. The MWCNT GOx nano bioconjugates showed about 5–7 times higher enzyme activity and exhibited significantly improved stability during storage in comparison to the free enzyme. The optimum pH and temperature for GOx-MWCNT bioconjugate with two different functionalized MWCNTs (MWCNT-COOH and MWCNT-NH₂) were found to be different by the Taguchi experimental design (TED) and confirmed by experimental studies. GOx-MWCNT nano bioconjugate with MWCNT-NH₂ specifically exhibits higher enzyme activity at pH 7, 30^°C, compared to immobilization of GOx-MWCNT nano bioconjugate with MWCNT-COOH. This study shows that amine and carboxyl nano bioconjugates have different optimum conditions for operations. The optimum conditions could be judiciously used for one-step fabrication of bioelectrode. High catalytic efficiency, enhanced stability, and lyophilized form also offers practical advantage for storage and commercial distribution under refrigerated conditions and can replace use of free enzyme in various industries.</p>