Abstract: To address the current challenges of limited sensitivity and inadequate stability in enzyme-free glucose sensors, this study constructed a high-performance enzyme-free glucose sensing electrode through a bimetallic synergistic modification strategy. Titanium dioxide nanotube arrays (TNTs) were fabricated via anodic oxidation. Nickel nanoparticles (Ni NPs) and gold nanoparticles (Au NPs) were sequentially deposited on TNTs through pulse electrodeposition and hydrothermal methods, yielding the Ni-Au@TNTs composite. Electrochemical tests revealed that the Ni-Au@TNTs electrode demonstrated a sensitivity of 3207 μA·L/(mmol·cm²) within 10 μmol/L - 2.17 mmol/L, a low detection limit of 0.74 μmol/L (S/N = 3), a response time below 1 s, along with excellent anti-interference capability and long-term stability. In clinical serum testing, the glucose concentration measured by the Ni-Au@TNTs electrode closely matched the hospital's reference values, with a relative standard deviation (RSD) of 4.54%. The study demonstrates that the synergistic effect between Ni and Au nanoparticles significantly enhances catalytic activity. The high specific surface area of TNTs and optimized electronic structure at the bimetallic interface collectively facilitate rapid glucose oxidation. This work provides a new strategy for developing efficient and stable enzyme-free glucose sensors.