In this work, the β-cyclodextrins (βCD) grafted on magnetite@polynorepinephrine (Fe3O4@PNE) nanomaterial with glucose oxidase (GOx) from Aspergillus niger was presented. The electroactive nanoplatform was used to construct rapid response and long-live time biosensor for qualitative and quantitative glucose determination. The nanomaterial was deposited on the screen-printed electrode (SPE) and integrated with the potentiostat in tandem with a portable devices. The methodology may affect its relatively low unit cost, miniaturization aspect, and electrode system integrity. The potential usage is intended for advanced diabetes care with a focus on the point-of-care testing idea. The cyclic voltammetry and amperometry were used for electrochemical characterization. The presented SPE/Fe3O4@PNE@βCD-GOx biosensor enabled measurements in a wide range of concentrations (0.1–30.0 mM), an enhanced sensitivity (204.82 µA mM− 1 cm− 2), a low limit of detection (3.2 µM), and a rapid response (2.6 s). Moreover, the proposed sensor achieved long-term stability, up to 11 months. Testing on real samples (human blood, human serum, infusion fluids) showed recovery in range from 95.5 to 98.6%. The outcomes demonstrated that this biosensor has great potential for use in determining the amount of glucose in a biological fluids and commercial products. The novelty of this work would largely consist of the possibility of qualitative and quantitative measurements of glucose in real human samples with a long time stability. This portable system enables mobile diagnostics tests including point-of-care testing idea. Due to the applied β-cyclodextrins on the surface of the novel polynorepinephrine biopolymer coating, selectivity, stability, and sensitivity were improved.Graphical