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• Electrochemical glucose biosensor based on metal-free N-doped carbon material obtained from polyaniline. • High glucose sensitivity 23.57 ± 1.77 μA cm−2 mM−1 and linear working range 5 µM- 5 mM at −0.4 V vs. Ag/agcl (3 M KCl). • Glucose detection and quantification in commercial sugary drink and human urine sample have been performed. An enzymatic metal-free electrochemical biosensor based on immobilized glucose oxidase on a N-doped porous carbon material has been studied for the detection of glucose in different real samples. The N-doped carbon material, which contains quaternary nitrogen groups, has been obtained by heat treatment of polyaniline. These functionalities promote the electrocatalytic activity to oxygen reduction reaction. The electrochemical biosensor presents a remarkable electrochemical activity towards oxygen reduction reaction, being able to detect changes in oxygen concentration generated by the enzymatic oxidation of glucose, allowing the indirect detection of this compound in phosphate buffered solution (0.1 M PB solution, pH=7.2). The metal-free electrochemical biosensor shows an outstanding performance, with a linear detection range between 5 µM and 5 mM and a high sensitivity of 23.57±1.77 µA mM−1 cm−2 at -0.4 V vs. Ag/AgCl. At these conditions, the addition of ascorbic acid, dopamine, uric acid and l -gluconic acid does not interfere with glucose determination. The method has been successfully applied to human urine and commercial sugary drink samples, without significant matrix effects, providing an alternative for glucose detection. Image, graphical abstract [ABSTRACT FROM AUTHOR]