Abstract: Objective：To establish a sandwich ELISA for detecting thioredoxin reductase(TR) GliT of Aspergillus fumigatus, and try to detect GliT in the supernatant of different fungi cultures. Methods：HRP-conjugated rat anti-TR monoclonal antibody and anti-TR goat polyclonal antibody was used as the detector antibody and coating antibody respectively. Preliminary checkerboard titration experiments were performed to determine the optimal concentration of the coating antibodies and the detecting antibodies. The performance parameters of the sandwich ELISA including precision, specificity and limit of detection were evaluated. This assay was applied to determine TR levels in the supernatant of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Candida albicans, Candida tropicalis and Candida glabrata cultures. Results：The optimal concentration of coating antibody was 1 μg/mL and the dilution of the detecting antibody was 1∶3 000. The limit of detection was 2.25 ng/mL. The intra and inter-coefficient of variation was 8.57%, 12.69% and 6.73%, 10.45%, at the TR concentration of 5 ng/mL and 20 ng/mL, respectively. The sandwich ELISA could specifically detectedTR in Aspergillus fumigatus cultrue after incubation at 37 ℃ for 24 h, and no cross reactivity to Aspergillus flavus, Aspergillus niger, Candida albicans, Candida tropicalis or Candida glabrata was found. The level of TR in supernatant of fungi cultures increased with growth of hyphae. Conclusion：A specific and sensitive sandwich ELISA for detection of Aspergillus fumigatus antigen TR was developed. This method might be valuable in diagnosis of invasive aspergillosis.