We investigated the biosorption characteristics of thallium (I) ions using dead biomass of Pseudomonas fluorescens strains as biosorbents. The biosorbents were characterized by Fourier transform infrared spectroscopy (FT-IR) and a scanning electron microscope (SEM). The effects of different environmental factors such as initial Tl concentration, initial solution pH, biomass dosage, and contact time were evaluated. The maximum adsorption capacity was found to be 93.76 mg/g at an optimum initial pH of 5.0, a contact time of 60 min, a biomass of 0.5 g/L, and an initial Tl concentration of 50 μg/mL. The biosorption process can be well defined by the Langmuir isotherm (R² = 0.9967). The biosorption kinetics were better described by the pseudo second-order model (R² = 0.9950) than the pseudo first-order one. The analysis of (FT-IR) indicates that the main functional groups responsible for adsorption of Tl (I) were hydroxyl, carboxyl, and amino groups. SEM analysis verifies an obvious surface morphology change of adsorbed biomass. The results presented in this study show that the Pseudomonas fluorescens could be an effective, low-cost, and environmentally friendly biosorbent for removing Tl (I) from aqueous solution.