Abstract: This study aims to elucidate the temporal dynamics and variation characteristics of nerolidol production in Stropharia rugosoannulata, and to further improve the research system for its volatile components. A method based on headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used for the qualitative and quantitative determination of nerolidol in S. rugosoannulata. The adsorption conditions were optimized, the effects of growth stage, cultivation mode and storage process on nerolidol content were systematically investigated. The cultivar ‘Jinqiu Nomber 18’ cultivated both in greenhouse and under forest was selected as plant material, and the content changes of nerolidol during different growth stages and storage processes were analyzed by optimizing adsorption temperature and time and by validating the analytical method. The results showed that nerolidol in S. rugosoannulata, existed predominantly as the trans-isomer with a standard curve exhibiting R² of 0.996, indicating high reliability of the method, the optimal adsorption conditions were 90 ℃ for 40 minutes. During the growth period, the nerolidol content reached its peak at 48 h after fruiting and then declined gradually, the content in greenhouse-cultivated samples was significantly higher than that in under-forest cultivated samples, while the content throughout storage continuously decreased. This study fills a research gap regarding the content and dynamic changes of nerolidol in S. rugosoannulata, providing theoretical support for the regulation of its flavor quality and optimization of preservation technology, and laying a foundation for its potential application in food flavoring and daily-use fragrance and other fields.