This work presents the development of a membraneless single-enzyme biofuel cell powered by glucose (GBFC). The GBFC biocathode is based on a graphite rod electrode (GRE) coated with a layer of Prussian blue (PB) nanoparticles entrapped into poly(pyrrole-2-carboxylic acid) (PPCA) shell, an additional layer of PPCA, and covalently to polymer linked glucose oxidase (GOx). The bioanode is based on GRE modified with a nanobiocomposite composed of poly(1,10-phenanthroline-5,6-dione), gold nanoparticles entrapped in a PPCA shell, and GOx linked by an amide bond to polymer. The operation of the developed single-enzyme GBFC is based on GOx-catalysed oxidation of glucose on both the bioanode and biocathode and reduction of H₂O₂ electrocatalysed by PB on the biocathode. The GBFC operated in O₂-saturated buffer medium pH 6.0 containing glucose. An open-circuit voltage (OCV) of 646 mV, a maximum power density of 10.94 μW/cm² and a current density of 60.52 μA/cm² at 40 mM glucose were determined. OCV and current density were directly proportional to glucose concentration in 0.01–10.00 mM and 0.05–124.00 mM concentration ranges, respectively. In addition, GBFC had good operational stability and retained more than 90% of the initial OCV after 36 days.