Background: Acori Tatarinowii Rhizome (ATR) is a well-recognized Chinese herbal medicine prescribed to treat neurological disorders. The essential oil (ATEO) is considered as the active fraction of ATR and the content of ATEO is used as the only indicator for ATR content determination. The quality of ATEO varies widely due to region difference; however, little is known about how to study ATEO quality chemically and biologically in response to region difference. Thus, it is of great importance to identify volatile active components in ATEO to conduct quality study. In this study, we analyzed ATEO from different regions in China using chemical component analysis combined with biological activity evaluation. Methods: GC-MS was used to obtain different volatile component profiles of ATEO and significantly changed volatile components were screened out. The neuroprotective activities of ATEO, including anti-oxidation, antiinflammation and neurotrophic functions, were revealed in C6 glioma cells. The correlation study between the bioactivities and the components was performed. Results: 57 volatile components, including terpenoids, phenylpropanoids, aromatic compounds, and other aliphatic compounds, were identified. 8 volatile components (β-asarone, cis-methyl isoeugenol, γ-asarone, methyleugenol, calarene, longifolene, β-caryophyllene and caryophyllene oxide) from ATEO were significantly changed due to region difference and 2 of them (β-asarone and γ-asarone) showed strong correlation with neuroprotective activities. Conclusions: Our results reveal that ATEO from different regions in China show great changes in chemical composition and biological activity. Moreover, phenylpropanoids (β-asarone and γ-asarone) present strong correlation with the bioactivities, which are considered as volatile active components in ATEO. The findings will be useful for the development of quality study of ATEO.