MXenes-Ti3C2Tx, based on their versatile surface characteristics, has rapidly advanced as an interactive substrate to develop electrochemical sensors for clinical applications. Herein, Ni embedded Ti3C2Tx, (MX-Ni) composites were prepared using a self-assembly approach where Ti3C2Tx, 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, Ti3C2Tx and Ti3C2Tx-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 Ti3C2Tx enabled sensitive signal output in wide detection ranges of 0.001 to 0.003 mu M and 0.0035 to 0.017 mu M and a detection sensitivity down to 0.12 mu M 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.