In in vitro experiments, the possibility of using a luminescent system extracted from the luminous fungus Armillaria borealis has been shown to detect and determine the concentration of hispidin. A linear dependence of the luminescent response on the content of hispidin in solutions in the concentration range of 5.4 × 10-5-1.4 × 10-2 µM was detected. The stability of the enzyme system and the high sensitivity of the bioluminescent reaction allows carrying out multiple measurements with the analyte detection limit of 1.3 × 10-11 g. The obtained results show the prospects of creating a rapid bioluminescent method for the analysis of medical substances or extracts from various biological objects for the presence of hispidin.