The essential oil was isolated using the ultrasound method from the root of Rehmannia glutinosa Libosch root with a yield of 0.47 % (w/w) by orthogonal matrix method. This yield of essential oil with steam distillation after the ultrasound method (EOU) is 2.03 times than that without ultrasound-assisted isolation (EO). The essential oil samples with and without the ultrasound method were analyzed by GC-MS. Total forty-six components in EOU were identified. The major compounds in the EOU and EO were found to be identical namely methyl palmitate, ethyl oleate, furfural, and methyl linoleate. Antioxidant activity of the samples were determined by two different test systems namely hydroxyl radical scavenging activity assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay. EOU showed higher antioxidant properties than EO, probably due to their different compositions.