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Background: β-D-glucan detection is a useful diagnostic indicator of invasive mycosis. However, the differences among the commercial β-D-glucan assays are unclear. Herein, we explored the diagnostic value of various β-D-glucan assay reagents. Methods: This prospective observational study involved 175 eligible patients suspected to have fungal infections. For all participants, culture examinations were conducted with specimens obtained from the infected site (or blood culture), and β-Dglucan was measured using three commercial kits: Wako β-glucan test (Wako), Fungitech G-test MKII “Nissui” (MKII), and Fungitech G-test ES “Nissui” (ES). Results: A total of 163 participants were included. Among them, 32 cases of invasive mycosis, 34 cases with mycotic colonization infection, and 97 cases with non-fungal infections were confirmed. Regarding the diagnostic value of the commercial kits for invasive mycosis, the areas under the receiver operating characteristic curves were > 0.8 for all the agents. However, on the basis of the cut-off value set by the manufacturer, the sensitivity and specificity of the three kits for definitive invasive infection were 80.0% and 90.6% with Wako, 80.0% and 80.7% with MKII, and 86.7% and 71.8% with ES, respectively. Moreover, the rate of false-positive β-D-glucan elevation detection in patients with negative fungal culture was 9.3% with Wako, 18.6% with MKII, and 23.7% with ES. Conclusion: Despite the high diagnostic value of β-D-glucan detection in invasive fungal infections, caution should be exercised in interpreting the value of the assay reagents. [ABSTRACT FROM AUTHOR]