Abstract: The study proves that the oleander plant, Nerium indicum Mill., and its extracts has stronger toxic effects to many insect pests, and displays a greater role in insect biological control. In order to rationally development and utilization of N. indicum plants, the safety of aquatic organisms and toxicological evaluation of mammalian animals were further evaluated. In the paper, ethanol crude extracts from N. indicum leaves were extracted by using the Soxhlet extraction method, and ethanol fine extracts were further separated by the method of extraction and concentration of chloroform, silica gel column chromatography, and the result of identification was cardiac glycosides. Toxicology of ethanol crude extracts on experimental animals was evaluated by entrusting Zhejiang Academy of Medical Science, acute and chronic toxicity of ethanol crude extracts and fine extracts (cardiac glycosides component) to Brachydanio rerio were studied by using static method. The results showed that extraction rate of ethanol crude extracts and fine extracts from N. indicum leaves was 45% and 0.25%, respectively. Oral and percutaneous toxicity of ethanol crude extracts to rats was on the low toxicity level, skin and eye irritation to rabbits belong to non-stimulation, hypersensitive reaction to guinea pig belong to weak sensitization. It indicated that ethanol crude extracts from N. indicum leaves was very safe to mammalian. In the acute toxic experiment, obvious positive correlation between the mortality of zebra fish and treatment concentration and treatment time of extracts was found. The higher treatment concentration was, the higher dead rate of the fish was; the longer treatment time under the same concentration was, the higher mortality of zebra fish was. When treated with 24 mg/L of ethanol crude extracts for 6h, 12h, 24h and 48h, the mortality of B. rerio were 26.67%, 60%, 91.11% and 95.56%, respectively. When treated with 0.5 mg/L of ethanol fine extracts for 12h, 24h, 48h, 72h and 96h, the mortality of the fish were 6.25%, 33.33%, 52.08%, 54.17% and 60.42%, respectively. When treated for 96h by 24 mg/L of ethanol crude extracts, or treated for 48h by 32 mg/L of ethanol crude extracts, or treated for 24h by 1 mg/L of ethanol fine extracts, the zebra fishes were all dead. In the chronic toxic experiment, the mortality of the zebra fish was about from 12% to 20% when treated for 28 days by 3.33-10.0 mg/L of ethanol crude extracts. After treated by ethanol crude extracts, medium lethal concentration of acute toxicity (LC50) to B. rerio was 12.52 mg/L (96h) and LC50 of chronic toxicity was 199.51 mg/L (28d). After treated by ethanol fine extracts, medium lethal concentration of acute toxicity (LC50) of B. rerio was 0.46 mg/L (96h). According to the toxicity grading criteria of pesticides to fishes from chemical pesticides environment safety evaluation test criterion, toxicity of ethanol crude extracts from N. indicum leaves to B. rerio was low toxic and very safe, while ethanol fine extracts to the zebra fish was high toxic and unsafe.