Objective To explore the application effect of tracer methodology in the investigation of microbial contamination of hemodialysis fluid. Methods Tracer methodology was used to perform on-site tracing for the unqualified events in microbial monitoring on hemodialysis fluid, relevant data were collected and analyzed with quality management tool, the existing root causes were found out, special rectification was made, disinfection and isolation system in hemodialysis room was improved, so as to promote continuous improvement of management quality of healthcare-associated infection and ensure safety of hemodialysis. Results Before the implementation of tracer system, 125 hemodialysis fluid and relevant specimens were taken, 80 of which were qualified, qualified rate was 64.0%. After comprehensive rectification on disinfection, dialysis fluid was continuously monitored and sampled for half a year, 125 specimens were taken, 116 were qualified, qualified rate was 92.8%. There was significant diffe-rence in the qualified rate of hemodialysis fluid and related specimens before and after improvement (P<0.05). Conclusion Tracer methodology is a very effective quality management tool, which can be used for investigation on adverse events of dialysis fluid microbial contamination and continuous quality improvement, and can be promoted and applied in the actual practice of healthcare-associated infection management.