Taking a third class a hospital in Nanjing as the research object, this paper discusses the effect of single disease payment mode from the investigation of the overall hospitalization cost and disease cost before and after the implementation of single disease payment mode, and provides reference suggestions for improving the reform of single disease payment mode and medical insurance payment mode. In this paper, retrospective analysis was carried out to select the cases and 10 sample diseases from November 2015 to December 2021 in a third class A hospital in Nanjing, and comparative analysis was made on the relevant indicators before and after payment of single disease in the hospital. The results showed that after the implementation of single disease payment, the average length of stay was shortened by 1.21 days on average, the average total medical cost and drug cost decreased, but the overall fund, project cost, material cost and anesthesia cost increased instead of decreasing, with statistical significance (P<0.05). The single-disease payment system slows down the growth of medical expenses to a certain extent and promotes the standardization of medical service behavior. We should strengthen the control of single disease fees, actively explore the transition from single disease payment to DRG payment, and improve the payment system in which multiple payment methods coexist.