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In this study, eco-friendly and disposable paper-based membraneless microfluidic enzymatic fuel cells (EFCs) were developed without any mediators to reduce the toxicity and cost of EFCs. Glucose oxidase and laccase were immobilized on multi-walled carbon nanotube electrodes to catalyze the redox reaction of glucose and oxygen. Micromachining techniques well-suited for mass production were used to precisely fabricate micro-scale Y-shaped and cross-shaped EFCs. Experimental measurements showed that the concentration of glucose in the fuel solution affects the cell performance, which occurs because the flow speed of the fuel stream decreases as the concentration of glucose increases. The highest performance of power density (104.2 ± 3.35 μW cm−2) and current density (615.6 ± 3.14 μA cm−2) were obtained with the Y-shaped channel configuration at a glucose concentration of 100 mM. This performance is the best of all paper-based single EFCs reported to date. The new paper-based co-laminar flow mediatorless EFC exhibits strong potential to power miniaturized and portable on-site diagnostic devices. • The first mediatorless paper-based co-laminar flow enzymatic fuel cell was developed. • A glucose biofuel cell was micromachined to be precise and mass-producible. • The microchannel of the biofuel cell was designed with Y-shaped and cross-shaped structures. • Electrolyte flow speed decreased with the rise of glucose concentration in the electrolyte. • The proposed fuel cell shows better performance than enzymatic fuel cells with a mediator. [ABSTRACT FROM AUTHOR]