The overall objective of this study is to understand the mechanisms by which geochemical controls on tetracycline speciation govern induction and expression of microbial antibiotic resistance in environmentally relevant soil and water systems. Tetracyclines are a class of antimicrobials that have been extensively used to improve human and veterinary health. A large portion of tetracyclines used by humans and animals are released into the environment, raising concerns of the potential risks to ecosystems and human health. Tetracycline generally manifests several species with the fractional distributions of each species depending on the pH-pKa relationship in aqueous phase. These species could interact with geochemical cations (e.g., Ca2+ and Mg2+) to form multiple complexes in water. In this study, we used E. coli MC4100/pTGM whole-cell biosensor as an effective tool to investigate the bioavailability of tetracycline in the solution phase under varying conditions (pH, type of cations, cation concentration). The results revealed that the activities of the biosensor respond in a linear manner to tetracycline intracellular concentrations which were measured by liquid chromatography coupled to tandem mass spectrometer. At a higher pH (e.g., pH = 8.0) less amount of tetracycline entered the cells demonstrating a reduced bioavailability as compared to the tetracycline in the solutions at pH of 6.0 and 7.0. The presence of Mg2+ and Ca2+ in the solution could bind to tetracycline and form metal-tetracycline complexes, hence reducing the diffusion of tetracycline into the cells. Increased concentrations of Mg2+ and Ca2+ in solution enhanced such complexation and decreased the bioavailability. Among the species of tetracycline present in solution including free tetracyclines and metal-complexed species, we identified zwitterions of tetracycline as the species most ready to penetrate the cell membrane and cause the response of E. coli whole-cell reporter. Understanding the relationship between tetracycline speciation and bioavailability to bacteria is an essential step to assess their exposure and risks for the tetracyclines present in the environment.