Warming and copper (Cu) toxicity are two key abiotic stressors that strongly affect cell growth, photosynthetic rate, and metabolism in microalgae. In this study, a freshwater chlorophyte, Scenedesmus quadricauda, was exposed to various concentrations of copper sulfate (300, 600, and 1000 μM nominal concentrations of CuSO4·5H2O) at 25 and 35 °C. The changes in cell density, photosynthetic parameters, in vivo absorption spectra, reactive oxygen species (ROS) levels, and metabolic profile were analyzed. The effects of copper toxicity on the physiology and biochemistry of microalgae were highly dependent on water temperature. The interactive effects of both stressors induced significant impact on the photosynthetic parameters such as maximum quantum yield (Fv/Fm), saturation irradiance (Ek), and non-photochemical quenching (NPQ). Temperature induced significant impact on cell density, Ek and NPQ, while the Cu toxicity significantly affected the Fv/Fm and NPQ. Changes in the in vivo absorption spectra and high levels of reactive oxygen species (ROS) were observed across different treatments. Overall, S. quadricauda adapted to the two abiotic stresses via NPQ and metabolic restructuring. Key metabolites including glycine, proline, hexadecanoic acid, propanoic acid, octadecanoic acid, galactose, lactose, and sucrose were involved in the microalgal response. The synergistic effects of temperature and Cu stresses on microalgae might affect community tolerance and species distribution in the long run.