【Objective】The potential impact of biochar on key soil animals, especially earthworms, is an important aspect in evaluating its environmental safety. Although previous studies have confirmed the toxic effects of biochar on earthworms, there is still a lack of systematic understanding of how preparation conditions regulate its toxicity and the specific sources of toxicity. Thus, this restricts the screening and application of low-risk biochar. 【Method】The effects of biomass sources (rice straw and perishable waste) and pyrolysis temperature (350 ℃, 500 ℃, and 650 ℃) on the toxicity of biochar to earthworms were systematically investigated. Also, the toxicity differences between biochar and its extract solution were compared. Moreover, the common Eisenia fetida was used as the test subject, and multiple indicators were employed, such as individual survival rate, enzyme activity, histopathological observation, and transcriptomics, to evaluate the effect of biochar on the earthworm’s survival. 【Result】The main results indicated that: (1) The toxic effects of biochar on earthworms exhibit significant feedstock specificity and dose dependence. At an exposure dose of 2.5 g·kg-1, perishable waste biochar (PWB) significantly reduced the relative survival rate of earthworms, while the same dose of rice straw biochar (RSB) had no significant effect; when the dose increased to 5 g·kg-1, both types of biochar showed obvious lethal effects on earthworms. (2) The response of the oxidative stress system showed that biochar stimulated the antioxidant defense mechanism of earthworms. Moreover, as the pyrolysis temperature increased, the oxidative stress in the earthworms showed a decreasing trend. The integrated biomarker response further indicated that the ecological risk of PWB was higher than that of RSB, and the toxicity risk of biochar pyrolyzed at a lower temperature was stronger. (3) Histopathological analysis indicated that biochar can induce damage to the epidermis and midgut of earthworms, with the damage caused by PWB being more severe. Transcriptomics revealed that several key physiological pathways such as protein digestion and absorption, ECM-receptor interaction, and vitamin digestion and absorption in earthworms, were affected under the exposure to biochar, and PWB induced more differentially expressed genes. (4) Biochar extract solution exhibited toxic tendencies to earthworms in the soil at a dosage of 5 g·kg-1, while the toxicity was lower than that of the solid biochar. This indicated that both the chemical composition toxicity of its extract solution and the physical characteristic risks of its solid particles should be considered for a comprehensive assessment of the ecological safety of biochar. 【Conclusion】Based on the above results, the toxic effects of biochar on earthworms in the soil are influenced by multiple factors such as the feedstock, pyrolysis temperature, dosage, and components of biochar. Thus, this study provides an important reference for research aimed at better assessing the environmental risks of biochar and provides a theoretical basis for the safe application of biochar in soil.