Abstract: Objective：To establish a double-antigen sandwich enzyme-linked immunosorbent assay(ELISA) for detecting the specific antibodies against Candida albicans enolase(Eno) in human serum, and evaluate its clinical application. Methods：The recombinant Eno prepared by the genetic engineering technology was used as coating and enzymelabeled antigens. The reaction conditions of the established double-antigen sandwich ELISA were optimized by the chessboard titration, and its sensitivity, specificity and repeatability were evaluated. Then, the serum samples from 291 inpatients, including 114 patients with invasive candidiasis(IC), 82 with Candida colonization and 95 with bacterial infection, and 200 healthy volunteers were collected and detected by the sandwich ELISA, and the obtained results were compared with those of indirect ELISA established by our laboratory previously. Results：The working conditions of the established double-antigen sandwich ELISA were as follows: 0.5 μg/mL of coating antigen Eno, 1∶4 000 diluted of enzymelabeled antigen, PBS-T containing 50 g/L of dried skimmed milk as the blocking buffer, and 1:100 diluted of serum samples. The intra-and inter-assay coefficients of variation of the established sandwich ELISA for the high, middle and low concentrations of serum samples were 6.8%, 7.4%, 5.9% and 10.1%, 9.6%, 12.4%, respectively. The blocking rate of antiEno antibodies in serum by the recombinant Eno was 92.2%. The absorbance value of the cutoff determined by the Receiver Operating Characteristics(ROC) curve was 0.209. The sensitivity and specificity of the established sandwich ELISA for the diagnosis of IC were 81.6%(93/114) and 94.4%(356/377), respectively, and its sensitivity was higher than that of indirect ELISA(81.6% vs 76.1%). Conclusion：The established double-antigen sandwich ELISA for the detection of specific antibodies against Candida albicans Eno may be useful for the clinical diagnosis of IC.