3-Hydroxy-4,5-dimethyl-2(5H)-furanone (sotolone) and 3-amino-4,5-dimethyl-2(5H)-furanone, the postulated precursor of sotolone, were detected in hairy root cultures of Trigonella foenum-graecum (fenugreek) by GC-MS. The hairy root cultures in both conical flasks and airlift with mesh bioreactors were achieved from hypocotyl of seedling by infection with Agrobacterium rhizogenes. In flasks, the mathematical relationship between hairy root growth and conductivity was established and afterward used to evaluate the biomass evolution in bioreactor cultures due to the difficulty of obtaining direct biomass samples from the bioreactor. The GC-MS analyses of ethanolic extracts from hairy roots revealed the presence of two important compounds: sotolone (1.2% of the volatile fraction) and 3-amino-4,5-dimethyl-2(5H)-furanone (17% of the volatile fraction). These results point out that biotechnological production of sotolone in bioreactors is possible. Additionally, these hairy root cultures offer, for the first time, an excellent biological model to study the biosynthetic pathway of sotolone in fenugreek.