Objective:To establish HPLC fingerprint and content determination methods of Zilian oil, which will provide the scientific basis for the quality control and evaluation of Zilian oil. Methods:TLC was used for the qualitative identification of Arnebiae radix, Coptidis rhizoma and Borneolum syntheticum in oil. HPLC was adopted for to determine the fingerprints of different batches, and the content determination of β,β'-Dimethylacrylalkannin of Zilian oil was determined. Results:TLC showed that Arnebiae radix, Coptidis rhizoma and Borneolum syntheticum had clear spots and good specificity. A total of 13 common peaks of Zilian oil were identified by HPLC fingerprint analysis, and 2 characteristic peaks (Berberine hydrochloride and β,β'-Dimethylacrylalkannin) were identified, and the similarity of fingerprints between batches was ≥ 0.981. The linear range of β,β'-Dimethylacrylalkannin was 40.48-202.40 μg. Conclusions:The methods of TLC identification, fingerprint and content determination are simple, reliable and stable, which can be used for the quality control of Zilian oil.