The invention relates to a single-molecule mechanical method for measuring inhibition of a small-molecule drug on protein and nucleic acid interaction. The method includes: first, designing and constructing a monomolecular reactor based on a DNA hairpin structure, the stem portion of which comprises a motif interacting with protein, and the handle portion of which is labeled with digoxin and biotin by polymerase chain reaction; secondly, preparing protein interacting with the DNA; thirdly, fixing the DNA hairpin structure between a magnetic bead and a glass sheet, and carrying out a DNA melting experiment in a microfluidic reaction tank through a monomolecular mechanics method; and finally, systematically testing the influence of the concentration of the small-molecule drug on the meltingreaction of the DNA combined with the protein, drawing a drug dose-response curve, calculating a half inhibition concentration, and evaluating the drug effect of the small-molecule drug on the proteinand nucleic acid interaction. The invention belongs to the field of biomacromolecule interaction and monomolecular pharmacology, and develops a new method for directly measuring small-molecule drug inhibition on protein nucleic acid interaction for molecular biology research, drug development and pharmacological evaluation.