The invention relates to a molybdenum disulfide based sensor as well as a preparation method and an application thereof and belongs to a protein detection method in the biomedical field. According to the technical scheme, the method mainly comprises steps as follows: firstly, an MoS2-AuNP compound is prepared; a nucleotide aptamer of thrombin is synthesized; the synthesized nucleotide aptamer is added to an aqueous solution of MoS2-AuNP, Tween 80 is added, a mixed solution is obtained, reacts for 30 min at the room temperature, then is heated for 2.5 h in a water bath with the temperature of 70 DEG C and is centrifuged at the speed of 10,000 r for 15 min, and then, precipitates are dispersed in a 0.1 M PBS (phosphate buffer solution); and the sensor is prepared. The characteristic that MoS2-AuNP can quench fluorescence is sufficiently utilized, an FAM (carboxyfluorescein)-marked nucleotide aptamer sequence is combined with thrombin specificity, trace detection of thrombin can be realized in a high-sensitivity, rapid and low-cost manner through quenching and recovery of FAM fluorescence, and the actual LOD (limit of detection) of the MoS2-AuNP-nucleotide aptamer sensor to thrombin is increased to 0.4 pM due to addition of Tween 80.