The outbreak of the influenza virus (H1N1) has symptoms such as coughing, fever, and a sore throat, and due to its high contagious power, it is fatal to humans. To detect H1N1 precisely, the present study proposed an electrochemical biosensor composed of a multifunctional DNA four-way junction (4WJ) and carboxyl molybdenum disulfide (carboxyl-MoS2) hybrid material. The DNA 4WJ was constructed to have the hemagglutinin aptamer on the head group (recognition part); each of the two arms has four silver ions (signal amplification part), and the tail group has an amine group (anchor). This fabricated multifunctional DNA 4WJ can specifically and selectively bind to hemagglutinin. Moreover, the carboxyl-MoS2 provides an increase in the sensitivity of this biosensor. Carboxyl-MoS2 was immobilized using a linker on the electrode, followed by the immobilization of the multifunctional 4WJ on the electrode. The synthesis of carboxyl-MoS2 was confirmed by field emission scanning electron microscopy (FE-SEM), and the surface of the electrode was confirmed by atomic force microscopy. When H1N1 was placed in the immobilized electrode, the presence of H1N1 was confirmed by electrochemical analysis (cyclic voltammetry, electrochemical impedance spectroscopy). Through selectivity tests, it was also possible to determine whether this sensor responds specifically and selectively to H1N1. We confirmed that the biosensor showed a linear response to H1N1, and that H1N1 could be detected from 100 nM to 10 pM. Finally, clinical tests, in which hemagglutinin was diluted with human serum, showed a similar tendency to those diluted with water. This study showed that the multi-functional DNA 4WJ and carboxyl-MoS2 hybrid material can be applied to a electrochemical H1N1 biosensor.