The label-free detection of nucleic acids has attracted interest of scientists due to the fact that it is simple, fast and efficient. Herein, l-cysteine was electropolymerized on the molybdenum disulfide (MoS2) surface to form a stable and electroactive poly(l-cysteine)-functionalized molybdenum disulfide (Pl-Cys/MoS2) sensing interface. Taking microRNA-21 (miRNA-21) as an analytical model, a label-free electrochemical sensor was designed according to the properties of the Pl-Cys/MoS2 sensing interface. Experimental data exhibited that the designed electrochemical sensor exhibited excellent sensitivity, selectivity and stability towards miRNA-21 detection in buffer and real samples. This study offers a methodology to construct a label-free sensing interface by combining MoS2 nanosheets and electroactive molecules.