Abstract: OBJECTIVE To develop substitution of reference substance method(SRSM) for simultaneous determination of furanodiene, curdione and germacrone in the Zedoary Turmeric oil. METHODS The determination of furanodiene, curdione and germacrone were performed on a C₁₈ analytical column(4.6 mm×250 mm, 5 μm) with acetonitrile-water as mobile phase in a gradient elution program at a flow rate of 1.0 mL·min^-1. The column temperature was maintained at 30℃. The detection wavelength was 216 nm. Germacrone was used as the internal reference substance, RCFs and RRTs of furanodiene and curdione were calculated. The contenets of furanodiene and curdione were determined by both the external standard method(ESM) and SRSM. RESULTS Furanodiene, curdione and germacrone had a good linearity rangings respectively(R² ≥ 0.999 9). RCFs and RRTs of furanodiene and curdione were stable. There was no significant difference between the results of SRSM and ESM. CONCLUSION The developed SRSM is simple and stable, which can determine the contents above mentioned in the Zedoary Turmeric oil instead of the ESM.