The increase of copper oxide nanoparticle (CuO-NP) utilization in industry during recent years has resulted in their entry into aquatic ecosystems. In light of this fact, we have studied the toxicity of CuO-NPs at various concentrations on Chlorella vulgaris using an algal growth inhibition test (OECD201). Chlorella vulgaris was grown in positive Zander (Z-8 + N) media in a growth chamber. After reaching to the logarithmic growth phase, the algae were exposed to various concentrations of CuO-NPs at intervals of 24, 48 and 72 hours. Algal cell numbers were counted daily and the data were analyzed by Probit analysis. Some parameters such as: the effective concentration (EC10, EC50, EC90), no observed effect concentration (NOEC), specific growth rate (μ), doubling time (G) and percentage growth inhibition (I %) were calculated. The values of EC10 = 12.588, EC50 = 43.699, EC90 = 152.019 and NOEC = 4.3699 mg/L were obtained after 72 hours. The results showed significant differences between control and treatments at specified intervals of cell density and percentage of growth inhibition (P <0.05). In addition, chlorophyll and carotenoid content in treated cells were significantly lower compared to those in control samples; a decrease in the efficiency of photosynthesis is very probably a major mechanism in the reduced growth and viability of C. vulgaris exposed to CuO-NPs.