Polygonum multiflorum Thunb. has been used widely in East Asia in treatment of diseases associated with aging. However, there are many reports referred to the toxicity of P. multiflorum, especially for liver adverse reactions. The toxicity of it is caused by over dosage or by the herb itself remains unclear. The aim of this study was to study the toxicity of different extractions, components and constituents of P. multiflorum, which were assessed in zebrafish embryos. Firstly, the difference of extracting solvent to the toxicity of P. multiflorum was researched to probe the influence of usages to the safety of P. multiflorum. The toxicity of 70% EtOH extract is considerably higher than that of other extracts. Secondly, 70% EtOH extract was subjected to macroporous resin (DM-8) eluting with a gradient of water and EtOH (H2O, 25% EtOH, 40% EtOH and 95% EtOH) to give four components (A-D). The toxicity of the component (D) showed higher than the other components (A-C). Thus, the component (D) was taken more attentions to research. Lastly, study on the chemical constituents of the component (D), 27 compounds, including 7 anthraquinones (1-7), 8 stilbenes (8-15), 7 anthrones (16-22), 3 cinnamic acid amides (23-25), 2 naphthols (26-27) were isolated and assessed in zebrafish embryos. Compounds 1-3, 16-22 and 26-27 showed severe toxicity against the zebrafish embryos while other compounds, such as stilbenes, showed no obvious toxicity.