Two-dimensional (2D) transition metal dichalcogenides (TMDCs) have attracted tremendous research interests due to their exciting optical properties, large surface area, intercalatable morphologies and excellent electrochemically catalytic activity. Acting as the most typical member in TMDCs family, layer-dependent molybdenum disulfide (MoS2) with particular direct bandgap of 1.8 eV in monolayer has been widely applied in various biosensors with high sensitivity and selectivity. In this review, the preparation methods of MoS2, together with MoS2-based biosensors for detecting cells and biomolecules (such as glucose, DNA and antigens) would be summarized. In addition, the current challenges and future perspectives are outlined for the applications of biosensors based on 2D MoS2.