The present study aimed to establish new protocol for propagation via tissue culture techniques to observe the effect of plant growth regulators especially cytokinins, gibberellic acid and auxins with different concentrations on in vitro growth of Eriocephalus africanus L. for improving the potentiality of regeneration and secondary metabolites production and identification of the main active constituents of volatile oil by GC/MS. The results showed that, the best sterilization treatment was the shoot tip explants rinsed in a solution of clorox at 15% for 15 min was gave the highest values for survival percentage and plant strength 100% and 4.58, respectively also B₅ medium at full strength gave the best results in the both growth measurements. BAP at 2.00 mg/l recorded the highest values in survival percentage (93.33%), shootlet number/cluster (16.50) and shootlet strength (4.50), respectively. Using the high level from GA₃ (4.00 mg/l) in medium was more effective in the elongation of shootlets. In rooting stage B₅ medium supplemented with 0.50 mg/l IBA and 0.15% active charcoal was more effective for increasing root number/explant to 8.67 and root length to 5.78 cm. The chemical analysis for the volatile oils extracted from three samples (field, in vivo and in vitro) of Eriocephalus africanus L. by using GC/MS confirmed that, the total number of the constituents identified ranged from 30-34 compounds representing 79.67-99.41% of the total oil contents. Artemisia ketone (17.10-30.62%), bicyclogermacrene (4.14-15.56%), globulol (2.17-8.30%), allo-aromadendrene epoxide (0.51-9.00%), caryophyllene oxide (2.85-6.76%) and α-pinene (3.10-4.25%) were the main compounds.