Abstract: Background: Gynura procumbens was usually used as traditional medicine in Indonesia, such as inflammation diseases, diabetes, cancer and hipertency. Production of biomass and secondary metabolite of this plant have potential to fulfill raw materials in pharmaceutical industry but harvesting of roots is destructive for the plants. This study was conducted to enhance biomass and secondary metabolite of adventitious roots in temporary immersion bioreactors. Materials and Methods: Adventitious roots of G. procumbens were cultured on liquid MS medium supplemented with various concentration of sucrose (1, 3 and 5%) and various immersion frequency (15 min each 12 h; 5 min each 3 h). Cultures were maintained for 21 days and fresh weight, dry weight and secondary metabolite profile were measured at the end of culture. Secondary metabolites were analyzed by Thin Layer Chromatography (TLC), TLC-scanner and Gas Chromatography-Mass Spectrophotometry (GC-MS). Results: The highest increasing of adventitious roots biomass were achieved on MS medium supplemented with 5% sucrose and immersion frequency 15 min each 12 h. Detection by TLC resulted two spot with different Rf (Rf1 and Rf2). Detection of maximum wave length by TLC-scanner resulted spectrum with λmax of band II is 261-270 nm and shoulders band I is 302-314 nm. These wavelength range was suspected of isoflavone group were corresponding from flavonoid compounds. The GC-MS analyzed showed that all treatment including adventitious roots ex vitro have volatile compound, which were known as adipic acid and bis (2-ethylhexyl) ester. Conclusion: Biomass production and secondary metabolite of adventitious root could increased significantly in temporary immersion bioreactor, so this technology have potential to develop in large scale.