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MXenes-Ti₃C₂T<i>_x</i>, based on their versatile surface characteristics, has rapidly advanced as an interactive substrate to develop electrochemical sensors for clinical applications. Herein, Ni embedded Ti₃C₂T<i>_x</i> (MX−Ni) composites were prepared using a self-assembly approach where Ti₃C₂T<i>_x</i> sheets served as an interactive conductive substrate as well as a protective layer to nickel nanoparticles (Ni NPs), preventing their surface oxidation and aggregation. The composite displayed a cluster-like morphology with an intimate interfacial arrangement between Ni, Ti₃C₂T<i>_x</i> and Ti₃C₂T<i>_x</i>-derived TiO₂. The configuration of MX−Ni into an electrochemical sensor realized a robust cathodic reduction current against methylmalonic acid (MMA), a biomarker to vitamin B12 deficiency. The synergism of Ni NPs strong redox characteristics with conductive Ti₃C₂T<i>_x</i> enabled sensitive signal output in wide detection ranges of 0.001 to 0.003 µM and 0.0035 to 0.017 µM and a detection sensitivity down to 0.12 pM of MMA. Importantly, the sensor demonstrated high signal reproducibility and excellent operational capabilities for MMA in a complex biological matrix such as human urine samples.