A miniaturized paper-based microfluidic electrochemical enzymatic biosensing platform was developedand the effects of fluidic behaviors in paper substrate on electrochemical sensing were systemically inves-tigated. The biosensor is composed of an enzyme-immobilized pure cellulose paper pad, an enzymelessscreen-printed electrode (SPE) modified with platinum nanoparticles (PtNPs), and a pair of clampedacrylonitrile butadiene styrene (ABS) plastic holders to provide good alignment for stable signal sensing.The wicking rate of liquid sample in paper was predicted, using a two-dimensional Fickian-diffusionmodel, to be 1.0 × 10.2cm2/s, and was verified experimentally. Dip-coating was used to prepare theenzyme-modified paper pad (EPP), which is amenable for mass manufacturing. The EPP retained excel-lent hydrophilicity and mechanical properties, with even slightly improved tensile strength and breakstrain. No significant difference in voltammetric behaviors was observed between measurements madein bulk buffer solution and with different sample volumes applied to EPP beyond its saturation wick-ing volume. Glucose oxidase (GOx), an enzyme specific for glucose (Glc) substrate, was used as a modelenzyme and its enzymatic reaction product H2O2was detected by the enzymeless PtNPs-SPE in the pres-ence of ambient electron mediator O2. Consequently, Glc was detected with its concentration linearlydepending on H2O2oxidation current with sensitivity of 10.5 A mM-1cm-2and detection limit of 9.3 M(at S/N = 3). The biosensor can be quickly regenerated with memory effects removed by buffer additionsfor continuous real-time detection of multiple samples in one run for point-of-care purposes. This inte-grated platform is also inexpensive since the EPP is easily stored, and enzymeless PtNPs-SPEs can beused multiple times with different EPPs. The green and facile preparation in bulk, excellent mechanicalstrength, well-maintained enzyme activity, disposability, and good reproducibility and stability makeour paper-fluidic biosensor platform suitable for various real-time electrochemical bioassays withoutany external power for mixing, especially in resource-limited conditions.