The conformational bias of tetracycline was investigated in a series of room-temperature 1-alkyl-3-methyl-imidazolium ionic liquids. Spectroscopic techniques such as circular dichroism, absorbance and fluorescence suggested that the switch between the extended and twisted conformation of tetracycline might be controlled by the aggregation state of the ionic liquids, which could be easily assessed by the resonance light scattering of homologous sets of ionic liquids with a common anion.