A highly sensitive and selective voltammetric biosensing platform for the determination of inosine (INO) concentrations and further applications to biological sample solutions was developed. The biosensor configures a layer-by-layer (LbL) self-assembly of the positively charged chitosan (CS) and negatively charged purine nucleoside phosphorylase (PNP) on screen-printed carbon electrode (SPCE); PNP was chosen to improve the selectivity since it is INO selective while CS was used for the enzyme PNP loading. The electrocatalytic reaction of INO and PNP was studied using cyclic voltammetry (CV) and square wave voltammetry (SWV). Under the optimal condition with two-bilayer of the PNP and CS on the modified electrode (PNP CS)(2)-SPCE, a linear dynamic range from 2 to 90 mu M INO with a limit of detection of 0.3 mu M was achieved when using SWV method. Finally, the developed biosensing platform was applied to the detection of INO in both normal human serum sample and myocardial infarction (MI) patient serum sample in addition to INO dietary supplements. The sensing results of normal human and MI patient serum samples were further compared to those obtained from liquid chromatography-mass spectrometry (LC MS) and a commercial inosine assay kit.