We report a single-step solid-state assisted synthesis of hexagonal boron nitride (hBN) nanosheets and the fabrication of a hBN modified glassy carbon electrode (hBN/GCE) for label-free, ultra-selective sensing of β-carotene in human blood serum samplesviathe differential pulse voltammetry (DPV) technique. Physicochemical analysis of the as-synthesised hBN reveals the few-layered structure of the hBN nanosheets with the presence of the (002) planeviafield emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD). The as-fabricated sensor exhibited a wide linear range of detection from 5 pM to 5 μM with a low limit of detection (3σ) of 1.25 pM, and a sensitivity of 82.519 nA pM−1cm−2. The enhanced sensitivity can be attributed to the improved electrocatalytic properties of the few-layered hBN nanosheetsviathe defects in the structure, which facilitated a strong interaction between the β-carotene oxidase and the hBN/GCE surface at the electro-electrolyte interface. This negatively impacts the rate of the redox reaction of the Fe3+/Fe4+redox couple which in turn reduces the redox peak current. The sensor further exhibits excellent stability, reproducibility, and selectivity and is successful in detecting β-carotene in the simulated blood serum samples with an outstanding recovery percentage ranging from ∼106.88 to ∼98.22. The performance of the as-fabricated sensor proves that a hBN/GCE is a promising platform for potential health care monitoring. © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020.