The invention discloses KIM-1 active targeting gold nanoparticles, a preparation method, and an application thereof, which belongs to the technical field of medical materials. The gold nanoparticles are modified on the surface of the gold nanoparticles with serine as a targeting ligand, which has high affinity and specificity for the kidney injury molecule KIM1. The preparation of GS-AuNPs is first synthesized, and then it is dissolved in PBS. After pH adjustment, EDC and NHS are added, serine is added, the pH is adjusted, and the product is obtained by ultrafiltration and centrifugation. The particles can be used as a CT contrast agent to detect KIM-1 in the kidney, and can also cooperate with phosphatidylcholine-modified silver nanoparticles to quantify KIM-1 in the kidney by urine analysis to achieve early non-invasive diagnosis of acute kidney injury, with low toxicity and good biocompatibility.