<p>Superoxide anion (O₂^•−) plays a crucial role in various biological processes. However, its excess can induce oxidative stress, cause cell damage, and promote diseases. Achieving ultra-sensitive detection of O₂^•− remains a significant challenge. Herein, a nanocomposite that integrates two-dimensional (2D) CoNi metal-organic framework nanosheets (CoNi-MOF) aerogel with carbon nanofibers (CNFs) through robust interfacial interactions was designed and fabricated. This CoNi-MOF@CNFs nanocomposite is tailored for high-performance electrochemical sensing of O₂^•−. The 2D CoNi-MOF features a rich mesoporous structure, which provides a multitude of reactive active sites. Meanwhile, CNFs effectively accelerate the electron transfer rate. The synergistic interfacial interactions between CoNi-MOF and CNFs enable efficient collaborative catalysis of O₂^•−, significantly enhancing the electrochemical catalytic ability compared to each component individually. The CoNi-MOF@CNFs-based sensor exhibits a high sensitivity (341.97 µA µM^− 1 cm^− 2), a low detection limit (0.83 nM), and excellent stability (2400&#xa0;s). More importantly, this fabricated sensor enables real-time monitoring of O₂^•− released by living cells, demonstrating its potential for disease diagnosis, highlighting the critical role of interfacial engineering in boosting the sensing performance of electrochemical sensors.</p> Graphical abstract <p></p>