Electrochemical nonenzymatic glucose sensing meets the challenge of excellent selectivity and the minimum poisoning effect of the electrode. Bimetallic alloys based on Pt have distinct synergistic characteristics and offer remarkable anti-poisoning capability and electrocatalytic performance for sensitive and selective glucose sensing at low potential. Bimetallic PtAu alloy nanomaterials loaded into nanocarbon carrier (PtAu/C) were successfully fabricated. The morphology and structure of PtAu bimetallic alloy were observed. The voltammetric response of the PtAu/C modified electrodes towards glucose oxidation in NaOH revealed significant enhanced compared with Pt/C or Au/C. PtAu (1:1)/C with 0.4 wt% content of Pt was used for selective glucose detection at a low potential of −0.33 V (vs. SCE). Interfering substances revealed inert and ignorable analytical influence on glucose detection. Furthermore, DPV was applied for sensitive determination of glucose. A linear relationship was found between peak current and glucose concentration within the range of 0.01–10 mM with the limit of detection (LOD) of 3 μM. Validation in human serum samples indicated that the PtAu (1:1)/C (0.4 wt% Pt) modified electrode accurately detected glucose. The PtAu bimetallic alloy exhibited a bright prospect for the glucose analysis of real samples. •Bimetallic PtAu alloy with homogeneous distribution of Pt and Au was fabricated.•PtAu/C shows catalytic and anti-poisoning capability for nonenzymatic glucose sensing•Mechanism of enhanced capability of PtAu is due to its strong synergistic effect.•PtAu/C for glucose sensing at low potential shows advantage of anti-interference.•This strategy for glucose sensing is sensitive, selective, stable, and reproducible.