This study presents a new and efficient method for detecting miRNA-191 by utilizing Ti3C2(OH)2 MXene Nanosheets. MicroRNAs have been established as important indicators of various types of cancer and other medical conditions, highlighting the need for dependable miRNA identification techniques. The proposed approach in this study revolves around the development of an enzyme-free, simple, quick, and label-free fluorescence probe. The combination of ssDNA-modified silver nanoclusters and Ti3C2(OH)2 MXene nanosheets generates a turn-on platform for detecting miRNAs. The utilization of MXenes, a novel two-dimensional material, has generated significant interest among researchers due to their exceptional properties, including sensitivity enhancement, stability, and reproducibility in biological fluids. The study revealed that ssDNA-AgNCs interact with the MXene surface using π-π stacking, resulting in the adsorption and quenching of fluorescence in AgNCs. The presence of the target molecule (miRNA-191) leads to the formation of a probe/target complex, which the releases ssDNA-AgNCs and restores their fluorescence. This fluorescence recovery is utilized to measure the concentration of miRNA-191. The designed platform exhibited excellent analytical performance with a linear range of 0.1–100 nM and a limit of detection (LOD) of ∼65 pM. The developed bioassay offers several advantages, such as ease of operation, cost-effectiveness, rapid results, and good stability and specificity. Notably, the optical biosensor based on Ti3C2(OH)2 MXene represents the first instance of such technology being applied to detect miRNAs. Additionally, the bioassay displayed satisfactory performance when assessed with real samples.