Layered double hydroxides (LDHs), regarding their physical and structural properties, have different and wide applications industry and their increasing use may raise ecological and human health concerns. However, the potential toxicity mechanisms of LDHs in different organisms are still unclear. In the present work, after synthesizing of ZnFe-SO4 LDH and studying of its characterization by XRD, FT-IR, SEM, EDX-mapping, TEM and Raman, its toxicity in Tetradesmus obliquus was evaluated. According to experimental results, the growth of the algae and content of photosynthetic pigments were significantly decreased after treatment with 100 mg/L of ZnFe-SO4 LDH. The high dose exposure to the LDH also inhibited the activity of SOD and POD enzymes, possibly due to the LDH- catalyzed reactive oxygen species production. In addition, lipid peroxidation and the content of phenolic compounds, as no-enzymatic antioxidants were increased by enhancement of the LDH concentration. The rise of phenol, flavonoids and MDA contents could be regarded as some manifestations and responses to the toxic effects of the contaminant in the algae cells. The results provided a better understanding of the undesirable effects and toxicity of LDHs in aquatic organisms.