Abstract: Flavonoid is one type of bioactive compound in the various edible fungi of the food samples, particularly known for its antioxidant, anti-inflammatory, and anticancer properties. There is a great contribution to the nutritional and medicinal values. However, reliable, efficient, and reproducible detection is often required to quantify the flavonoids in the fungi. Flavonoid detection can be expected to be applied to edible fungi, particularly for quality control and functional evaluation in the food and pharmaceutical industries. In this study, a relatively universal spectrophotometric method was proposed to determine the flavonoid content in fungi. Firstly, the existing approaches to flavonoid determination were reviewed in similar fields. A full-wavelength spectral scanning was employed using the gathered information. Three commonly consumed species of edible fungi were selected: Ganoderma lucidum, Inonotus hispidus, and Pholiota adiposa, due to their known flavonoid content and biological activities. A comparison was then made to evaluate the linearity, precision, stability, and recovery rate of spiked samples. Various experimental conditions were adjusted for the optimization, including the reaction times, reagent concentrations, and wavelengths. In particular, the sodium nitrite-aluminum nitrate-sodium hydroxide (NaNO₂-Al(NO₃)₃-NaOH) colorimetric method was selected for further flavonoid analysis. The results revealed that superior performance was achieved to detect the flavonoid, in terms of sensitivity and specificity. Among them, the interference was effectively eliminated from the background absorption peaks of the sample matrix. Furthermore, the bioactive compounds were often presented in complex biological samples, such as edible fungi, leading to the potential interference with the accuracy of the flavonoid quantification. There was a strong linear relationship between the absorbance and the flavonoid concentration, with a high determination coefficient (R²) exceeding 0.99 in all cases. The reliable and accurate measurements were realized in the concentration range that is typically encountered in edible fungi samples. Additionally, excellent precision and stability were found suitable for routine analyses, particularly with the low relative standard deviations (RSD) in the repeatability tests. There were extremely low values of below 0.01 and 0.02 in the detection limit (LOD) and quantification limit (LOQ), respectively, indicating high sensitivity. Furthermore, the optimal performance was achieved in the detection wavelength of 507 nm. Both the LOD and LOQ reached their extreme values for the most accurate and sensitive detection of the flavonoid compounds. NaNO₂-Al(NO₃)₃-NaOH) colorimetric method was highly suitable for the quantification of the flavonoids in edible fungi. The excellent linearity, precision, stability, and recovery can be expected to serve as an effective and reliable tool for the flavonoid content after validation. This finding can provide a solid reference for future research on the bioactive components in edible fungi, including their nutrition and physiological activities. Valuable insights can be offered for food quality control, functional food development, and the potential health benefits in the field of edible fungi. Additionally, the successful application can facilitate the standardization and commercialization of flavonoid-rich edible fungi products in the food and nutraceutical industries, thus promoting their health-promoting properties.