© 2018, Springer-Verlag GmbH Germany, part of Springer Nature.The use of a central composite design (CCD) for the optimization of electrode surface composition and its application to develop an amperometric glucose biosensor as a model system are described. A five-level three-factorial CCD was applied to determine the optimum electrode surface composition for three critical variables: amounts of carboxylated multiwall carbon nanotubes (c-MWCNT), titanium dioxide nanoparticles (TiO2NP), and glucose oxidase (GOx). The statistical significance of the model and factors were evaluated using the variance analysis (ANOVA) at 95% of confidence level. The optimized electrode surface composition was used for the fabrication of the glucose biosensor. The resulting biosensor showed linear response to glucose from 2.0 × 10−5 to 1.9 × 10−3 M with a detection limit of 2.1 × 10−6 M and sensitivity of 168.5 μA mM−1 cm−2 under optimal experimental conditions. Analytical performance parameters of the biosensor were also compared with those obtained with the glucose biosensors fabricated using the electrode compositions optimized by conventional one factor-at-a-time method and 22 CCD (for c-MWCNT and TiO2NP amounts). The optimization of the critical variables, achieved by CDD, leads us to fabricate the glucose biosensor in the best electrode surface composition which was promoted by the improved analytical performance. The proposed biosensor was applied to the analysis of glucose in serum samples and the obtained results were well correlated with the results of reference method. [Figure not available: see fulltext.].