We report the engineering and synthesis of peptide nucleic acid-functionalized Ti3C2Tx MXene nanosheets as a novel transducing material for amplification-free, nanoparticle-free, and isothermal electrochemical detection of microRNA biomarkers. Through bio-orthogonal copper-free click chemistry, azido-modified MXene nanosheets are covalently functionalized with clickable peptide nucleic acid probes targetting prostate cancer biomarker hsa-miR-141. The platform demonstrates a wide dynamic range, single-nucleotide specificity, and 40 aM detection limit outperforming more complex, amplification-based methods. Its versatility, analytical performance, and stability under serum exposure highlight the immense potential of this biofunctionalized 2D nanomaterial in the next generation of amplification-free biosensors.