MicroRNAs (miRNAs), critical biomarkers of acute and chronic diseases, play key regulatory roles in many biological processes. As a result, robust assay platforms to enable an accurate and efficient detection of low-level miRNAs in complex biological samples are of great significance. In this work, a label-free and direct hybridization assay using molybdenum disulfide (MoS2) field-effect transistor (FET) biosensor has been developed for ultrasensitive detection of miRNA-155 as a breast cancer biomarker in human serum and cell-line samples. MoS2, the novel 2D layered material with excellent physical and chemical properties, was prepared through sequential solvent exchange method and was used as an active channel material. MoS2 was comprehensively characterized by spectroscopic and microscopic methods and it was applied for fabrication of FET device by drop-casting MoS2 flacks suspension onto the FET surface. MoS2 FET device showed a relatively low subthreshold swing of 48.10 mV/decade and a high mobility of 1.98 × 103 cm2 V−1 s−1. Subsequently, probe miRNA- 155 strands were immobilized on the surface of the MoS2 FET device. Under optimized conditions detection limit of 0.03 fM and concentration range 0.1 fM to 10 nM were achieved. The developed biosensor not only was capable to identification of fully matched versus one-base mismatch miRNA-155 sequence, but also it could detect target miRNA-155 in spiked real human serum and extracts from human breast cancer cell-line samples. This approach paves a way for label-free, early detection of miRNA as a biomarker in cancer diagnostics with very high sensitivity and good specificity, thus offering a significant potential for clinical application.