The adsorption of microcystin-LR (MCLR) by biochar has never been well understood. For the first time, the unconventional adsorption of hydrophilic MCLR on wood-based biochars was comprehensively investigated as a function of biochar properties, environmental temperature, solution pH, coexisting dissolved organic matter (DOM), and polar organic competitors. High-temperature-prepared biochar from 700°C (BC-700) and low-temperature-prepared biochar from 300°C (BC-300) were characterized with significantly different surface areas but similar alkaline nature. Despite a very low surface area, BC-300 exhibited very high adsorption capacity, which implies the important contribution of surface groups to biochar. MCLR adsorption on biochars was pH dependent and was strongly reduced by macromolecular DOM. Polycarboxylic aliphatic acids and 2-(2-hydroxyethyl) guanidinium cation, which are similar to specific structural groups in MCLR, exhibited an evident competitive effect. The results indicated that both carboxylic and guanidino groups of MCLR serve significant functions in MCLR adsorption to biochar. The adsorption mechanisms may be primarily related to the columbic attractions and the hydrogen bonding interactions between MCLR and biochar surface. In particular, the irreversible adsorption enhancement of MCLR was observed on BC-700, which suggests that biochar amendment can aid in immobilizing MCLR from water to sediment, thereby prolonging MCLR environmental fate in biocharamended sediment.