〔Abstract〕 Objective To investigate the value of real-time quantitative polymerase chain reaction (qRT-PCR), (1,3)-β-Dglucan test (G test), and galactomannan antigen test (GM test) in the diagnosis of invasive fungal infection. Methods A total of 147 patients with suspected invasive fungal infection who were treated in the First People's Hospital of Zhengzhou from January 2020 to June 2022 were selected as the research subjects, and the fungal culture method was used to identify the fungal type in the infection group. The samples of patients with suspected invasive fungal infection were detected by qRT-PCR, G test and GM test, respectively, and the value of qRT-PCR, G test and GM test in the diagnosis of invasive fungal infection were analyzed with fungal culture results as the standard. Results Among 147 patients with suspected invasive fungal infection, invasive fungi were detected in 86 cases (positive rate 58.5 %), including 21 cases of Candida albicans (24.4 %), 18 cases of Aspergillus (20.9 %), and 15 cases of Candida tropicalis (17.4 %), 12 cases of cryptococcus (13.9 %), 10 cases of Malassezia (11.6 %), and 10 cases of yeast (11.6 %). Among the 147 patients with suspected invasive fungal infection, 74 cases were positive by qRT-PCR, with a positive rate of 50.3 %; 63 cases were positive by G test, with a positive rate of 42.9 %; 55 cases were positive by GM test, with a positive rate of 37.4 %. The sensitivity of qRT-PCR in diagnosing invasive fungal infection was higher than that of G test and GM test, and the difference was statistically significant (P < 0.05). The specificity of G test for diagnosing invasive fungal infection was higher than that of qRT-PCR and GM test, but the difference was not statistically significant (P > 0.05). Conclusion The positive rate of qRT-PCR for invasive fungal infection was higher than that of G test and GM test. The sensitivity of qRT-PCR in diagnosing invasive fungal infection was higher than that of G test and GM test, and the specificity of G test in diagnosing invasive fungal infection was higher than that of qRT-PCR and GM test.