The invention discloses a pepsinogen I detection kit and a preparation method thereof. The pepsinogen I detection kit is composed of the following components: PGI antibody-coating magnetic microspheres, a PGI calibrator, a PGI antibody marker bound to lanthanides, an analytical buffer, a cleaning solution, an enhancement solution and an RFID card. Based on combination of the magnetic microspheresand a time-resolved immunoassay method, the drawbacks of long physical adsorption reaction time and slow detection result of an enzyme label plate are overcome, and the reaction time is greatly shortened; besides, the advantages of high accuracy, high sensitivity, strong specificity, wide linear range, and stable and convenient detection of a time-resolved detection technology can also be realized. The magnetic microspheres are coated with corresponding antigens or antibodies, due to the stereoscopic characteristic of the magnetic microspheres, the contact surface area of an immune response isgreatly increased, the detection time is greatly shortened, and further optimization is made for the traditional two-step method detection, pepsinogen I detection only requires a one-step method, anda result can be obtained within 20 minutes.