The 96 h acute toxic effects of nano-CuO (N-CuO), micro-CuO (M-CuO) and ²⁺ on Chlorella sp. were investigated in this paper. The results showed that toxicities decreased in an order of Cu²⁺＞N-CuO＞M-CuO. The 96 h EC₅₀ of Cu²⁺ on Chlorella sp. was 1.06 mg /L, and of N-CuO it was 74.61 mg /L, while no pronounced toxicity was observed when the concentration of M-CuO was lower than 160 mg/L. Further experiments were carried out in order to study the toxicity mechanism of nano-CuO on Chlorella sp.. The results of Cu²⁺ release from N-CuO showed less than 0.2 mg/L Cu2+ were released, so the release of Cu²⁺ was not responsible for the toxicity. Further experiments showed N-CuO inhibited formation of Chlorophyll A. Content of Chlorophyll A in the control group was 4.75 mg/10⁸ cells, while it declined to 2.89 mg/10⁸ cells for 160 mg/L N-CuO after 96 h, which indicated that N-CuO could inhibit photosynthesis of Chlorella sp.. Moreover, N-CuO condensed with algal cells. It affected the activity of SOD and POD, indicating that N-CuO could cause oxidant stress to Chlorella sp.. These may be the toxicity mechanism.