Thallium (T1) is a rare element of high toxicity. The increasing T1 in the environment can enter aquatic systems and pose risks to freshwater organisms. In this study, the acute toxic effects of the exposure of freshwater organisms from the Pearl River to T1 were determined. Tests were performed using zooplankton (Brachionus calyciflorus and Daphnia magna; 48 h exposure), macroinvertebrates (Bellamya aeruginosa and Corbicula fluminea; 96 h exposure), and freshwater fish (Cirrhinus molitorella, Ctenogobius giurinus, Ctenopharyngodon idellus, Misgurnus anguillicandatus, and Oreochromis niloticus; 96 h exposure). The 48 h LC50 of T1 to B. calyciflorus, D. magna and B. aeruginosa, were 0.23, 0.49 mg/L, respectively. The 96 h LC50 of T1 to C. fluminea, C. molitorella, C. giurinus, C. idellus, M. anguillicandatus, and O. niloticus were 0.69, 0.88, 9.5, 6.7, 3.0, 12.8, and 8.7 mg/L, respectively. The aquatic vertebrates showed more tolerant of T1 than the invertebrates from the Pearl River. The species sensitivity distribution and regression analysis showed that sigmoid regression fitted the distribution best, and the estimated acute hazard concentration endangering 5% of species (HC5) was 14.12 mu g/L. The maximum and continuous exposure concentrations of T1 for preventing T1 toxicity to freshwater ecosystems in China in the short and long term were estimated as 7.06 and 0.71 mu g/L, respectively. The results provide basic data for preventing risks posed by T1 to ecosystems in areas with similar aquatic fauna of less T1 toxicity data.