AbstractThis study demonstrates a miniature glucose/O2 biofuel cell (BFC) with a high tolerance against physiological level of ascorbic acid (AA) by immobilising ascorbate oxidase (AAox) on both the bioanode and the biocathode. Single‐walled carbon nanotube (SWNT)‐modified carbon fiber microelectrodes (CFMEs) are employed as the substrate electrode for the bioanode and biocathode. Glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) are used as the biocatalysts for the electro‐oxidation of glucose and for the electro‐reduction of oxygen, respectively. SWNTs are used as the support for the both, stably confining the electrocatalyst (i.e. polymerised methylene blue, polyMB) for the oxidation of NADH co‐factor for GDH and efficiently facilitating direct electrochemistry of the cathodic biocatalyst (i.e. BOD) for O2 reduction. The prepared micro‐sized GDH‐based bioanode and BOD‐based biocathode employed for the bioelectrocatalytic oxidation of glucose and reduction of oxygen, respectively, are further over‐coated with AAox to give a miniature glucose/O2 BFC with a high tolerance against AA. The maximum power density and the open circuit voltage (OCV) of the assembled glucose/O2 BFC are 52 μW cm–2 and 0.60 V, respectively. These values remain unchanged with the presence of AA in solution. In the human serum containing 10 mM NAD+ and under ambient air, the maximum power density and the OCV of the assembled glucose/O2 BFC with AAox immobilisation on both the bioanode and the biocathode are 35 μW cm–2 and 0.39 V, respectively. These values are remarkably larger than those of the glucose/O2 BFC without AAox immobilisation on both the bioanode and the biocathode. This study could offer a new route to the development of enzymatic BFCs with promising application in real biological systems.