The invention belongs to the technical field of nano-bio sensing, and particularly relates to a miRNA sensor based on MoS2/Ti3C2 and a preparation method thereof and a detection method thereof. An AuNPs adsorbed on the surface of MoS2/Ti3C2 is connected with a terminal modified RNA biomolecule to construct a RNA/Au NPs/MoS2/Ti3C2 bioprobe. A hydrothermal method is employed to synthesize a novel two-dimensional nanocomposite MoS2/Ti3C2, the feature of the specific surface area of MoS2/Ti3C2, the excellent electrical conductivity of Au NPs and the capacity of differential selection and identification of the miRNA of the terminal modified RNA biomolecule are employed to achieve amplification of the detection signals so as to construct a miRNA sensor based on MoS2/Ti3C2. The miRNA sensor based on MoS2/Ti3C2 and the preparation method thereof and the detection method thereof can achieve rapid, high- sensitivity and high-specificity detection for the miRNA so as to have huge application potential in the fields of tumor detection and molecular biology.