Biochemical biomarkers and bioassays, due to their assumed immediate response after acute exposure of the organism to the stressor, are useful tools to gauging anthropogenic impacts. The toxicity of TiO2-nanoparticles (TiO2-NPs) on the Glyphodes pyloalis Walker was assessed and the LC50 value obtained as 660.85 mg/L. The in vivo responses of G. pyloalis to sub-lethal concentrations of TiO2-NPs were surveyed by monitoring the activity of general esterases (EST), peroxidase (POD) and glutathione S-transferase (GST), as biochemical biomarkers. Activity of these biomarkers affected by exposure to TiO2-NPs and this could lead to the mortality or sub-lethal impacts. The effect of TiO2-NPs concentrations on the activity of these enzymes was correlated to the exposure time. The activity of EST and GST was significantly decreased compared to the control, after 24 h of treatments. By increasing exposure time, the expression of EST and GST was significantly increased. More POD expression was occurred at low concentrations (i.e. LC20 and LC30); however, at high concentrations, less POD activity obtained. It can be concluded that these enzymes are good early indicator of toxicity and in conjunction with acute toxicity studies allow adverse effects of TiO2-NPs to be predicted and managed.