BackgroudThe global prevalence of diabetes mellitus, a serious chronic disease with fatal consequences for millions annually, is of utmost concern. The development of efficient and simple devices for monitoring glucose levels is of utmost significance in managing diabetes. The advancement of nanotechnology has resulted in the indispensable utilization of advanced nanomaterials in high-performance glucose sensors. Modulating the morphology and intricate composition of transition metals represents a viable approach to exploit their structure/function correlation, thereby achieving optimal electrocatalytic performance of the synthesized catalysts. ResultsHerein, a sensitive and rapid Cu-encapsulated Cu2S@nitrogen-doped carbon (Cu@Cu2S@N–C) hollow nanocubes-functionalized microfluidic paper-based analytical device (μ-PAD) was fabricated. Through a delicate sacrificial template/interface technique and thermal decomposition, inter-connected hollow networks were formed to boost the active sites, and the carbon shell was coated to protect Cu from being oxidation. For application, the constructed μ-PAD is used for glucose sensing utilizing an origami automated sample pretreatment system enabled by a simple application of strong alkaline solution on wax paper. Under optimal circumstances, the Cu@Cu2S@N–C electrochemical biosensor exhibits broad detection range of 2–7500 μM (R2 = 0.996) with low detection limit of 0.16 μM (S/N = 3) and high sensitivity of 1996 μA mM−1 cm−2. Additionally, the constructed μ-PAD also exhibited excellent selectivity, stability, and reproducibility. SignificanceBy rationally designing the double-shell hollow nanostructure and introducing Cu-encapsulated inner layer, the synthesized Cu@Cu2S@N–C hollow nanocubes show large specific surface area, short diffusion channels, and high stability. The proposed origami μ-PAD has been successfully applied to serum samples without any additional sample preparation steps for glucose determination, offering a new perspective for early nonenzymatic glucose diagnosis.