Fungal rhinosinusitis (FRS) is commonly caused by various Aspergillus species (spp) and Mucorales fungi, and the treatment and prognosis of cases differ depending on the causative fungus. The present study describes a novel immunohistochemical method that has high sensitivity and specificity for distinguishing between these two types of fungi in patients with FRS. Three groups were included in the study. Group A included formalin-fixed paraffin-embedded blocks of 51 nasal tissue specimens of patients with FRS (27 Aspergillus spp and 24 Mucorales) that were continuously obtained from the Department of Pathology of Tongren Hospital in Beijing as the experimental group and 34 cultures (26 Aspergillus spp and 8 Mucorales) of FRS that were randomly selected from the bacterial laboratory of Tongren Hospital in Beijing to verify the staining results of the paraffin-embedded blocks. Formalin-fixed paraffin-embedded blocks of 10 esophageal cancer specimens were included in Group B as the positive control group. All specimens in Groups A and B were stained with interferon- (IFN-) antibody. Group C consisted of the same specimens as described in Group A, however, when performing the immunohistochemical assay, IFN- antibody was replaced by PBS and this served as the negative control group. The differences in IFN- immunohistochemical staining between Aspergillus spp and Mucorales were analyzed. Staining of IFN- in paraffin-embedded samples was positive in 92.6% (25/27) of specimens in which Aspergillus spp were the causative pathogen, which was significantly higher compared with specimens in which Mucorales was causative (P<0.001), with only 4.2% (1/24) of specimens staining positive for IFN-. Immunohistochemical staining of cell cultures was 100% positive for Aspergillus spp, whereas all Mucorales were negative. Thus, the results of the current study indicated that IFN- antibody immunohistochemical staining may be used as a novel diagnostic tool to distinguish between Aspergillus spp and Mucorales when identifying the causative agent in FRS, providing a useful supplementary test to the current immunohistochemical methods in the clinical diagnosis of FRS.