The present invention discloses a hypericin albumin nanoparticle-Escherichia coli serum antibody complex, which is obtained through adding mixed hypericin albumin nanoparticles and rabbit anti-Escherichia colis into ethanediol, irradiating them with carbon ion, setting them at a low temperature for 2 hours after irradiation, centrifuging them at a low temperature, and freezing and drying the precipitates. Its preparation method and application are also provided. The beneficial effects of the present invention are as follows: the present invention provides a hypericin albumin nanoparticle-Escherichia coli serum antibody complex and its preparation method and application, wherein through bacteriostatic test and clinical pharmacodynamic test, it is proved that the effects of target complex is greatly improved compared with that of the prior drug, because the Escherichia coli serum antibodies seek and capture Escherichia coli and the hypericin albumin nanoparticles inhibit and kill Escherichia coli, which precisely attacks Escherichia coli and strengthen the antibacterial effect of hypericin albumin nanoparticle on chicken focus location.