Objective The major aim of the present study were to prove that macromolecular impurities in polysorbate 80 for injection are a risk factor, and determine the main physical indexes of polysorbate 80 free of macromolecular substances. Methods The original polysorbate 80 for injection, with a theoretical molecular weight of 1 310 Da, was treated with ultra-filters to obtain component A (>100 kDa), B (10～100 kDa), and C (<10 kDa). The components were lyophilized to obtain their contents.Each component was prepared with normal saline at a concentration of 10%,anaphylactic and anaphylactoid reactions on guinea pigs that may be caused by the original polysorbate 80 and its components were recorded according to Chinese Pharmacopoeia of 2015th edition. Kunming mice were intravenously injected with them for 5 days administration and 5 days’ recovery to observe their organic toxicity. Results The proportion of substances with a molecular weight above 10 kDa in the original polysorbate 80 was above 85%. The original polysorbate 80 and its component A and B resulted in positive reactions in both anaphylactic and anaphylactoid guinea pigs, while component C didnt. In Kunming mice, component A and B caused an obvious retard of body weight increase in the 5 days’ recovery. The original polysorbate 80 and its components caused mild but significant toxicity in the liver, kidney, spleen, and lung at organ indexes both in 5 days administration and recovery. Their mild toxicity on the liver and kidney was verified by histological assay. However, toxicity caused by component C was much milder and more recoverable. The physicochemical constants of component C, including solubilization capacity, viscosity, density, absorption spectrum, pH, and particle size were improved. Conclusion All the results suggested that the main substances in polysorbate 80 for injection are macromolecular impurities that are an important risk factor to cause anaphylactoid reactions and organic toxicity, and macromolecular impurity removal improved its safety and physicochemical constants.