Objectives: Chronic fungal rhinosinusitis is a major health problem because of the large impact on quality of life and difficult medical treatment. Its prevalence is increasing. There is currently an opportunity to establish the diagnosis without invasive intervention by utilizing β-glucan, which is the largest component of fungal cell wall and neutrophil/eosinophil ratio (NER). We sought to discover the correlation between β-glucan level and the NER of paranasal sinuses mucosa and blood as a potential diagnostic marker for patients with chronic fungal rhinosinusitis. Methods: We conducted a cross-sectional study involving 20 subjects. Blood sampling and maxillary sinus surgery were performed, and fungal identification in the sinus mucosa was determined using the polymerase chain reaction. If a fungal species with β-glucan was found, then the examination was continued with the measurement of β-glucan by enzyme-linked immunosorbent assay, neutrophil, and eosinophil flow cytometry in sinus mucosa and blood. Results: Aspergillus flavus is the most commonly found fungus. All subjects passed the positive β-glucan limit (≥ 80 pg/mL) of the mucosal sample and only one subject had intermediate results (60–79 pg/mL) from their blood sample. Seventeen subjects had mucosal eosinophilic inflammation whereas the blood of 12 subjects revealed neutrophilic inflammation. There was no significant difference between the level of β-glucan in blood and mucosal sinus (p = 0.886), so that β-glucan examination of blood can describe β-glucan levels in paranasal sinuses. There was a significant difference between mucosal NER and blood (p < 0.001). β-glucan level with NER in both paranasal sinus mucosa and blood had no significant correlation (p > 0.050). Conclusions: β-glucan can be used to establish the diagnosis of chronic fungal rhinosinusitis. However, differential diagnosis of allergy or infection cannot be excluded by examination of blood NER. Still, it is hoped that the process of diagnosis can be obtained quickly and precisely without the need for invasive procedure although it requires more research, especially related to the diagnostic test.