The invention relates to a preparation method and an application of a monoclonal antibody. The preparation method comprises the following steps: 1, obtaining a B cell, enriching, and purifying; 2, binding a labeled antigen to a specific B cell; 3, obtaining an antigen specific B cell; 4, amplifying the variable region gene of an antibody; 5, constructing an expression vector; and 6, expressing a recombinant antibody and purifying: transfecting a 293T cell in the logarithmic phase by using purified expression vector DNA, centrifuging to remove cell fragments, and carrying out protein G column purification on the obtained centrifuge solution to obtain a recombinant monoclonal antibody solution. The preparation method substantially rises the concentration of the recombinant monoclonal antibody, reduces the cost, and is suitable for industrial large-scale production application. The obtained monoclonal antibody can specifically neutralize hepatitis C E1 antigens, and can be used for preparing drugs for preventing and treating hepatitis C.