In the field of nanoecotoxicology, very few reports have focused on biochemical changes in non-target organisms after nanoexposure. A less explored aquatic non-target crustacean, Moina macrocopa, was used in the present study to analyze toxicity effects of gold nanoparticles (AuNPs), an emerging nanomaterial. AuNPs was fabricated using tannic acid and were 29 ± 2 nm in size. The 48 h LC value of AuNPs was 14 ± 0.14 mg/L against M. macrocopa. The sub-lethal exposure of M. macrocopa juveniles to AuNPs (1.47 and 2.95 mg/L) decreased the activities of acetyl cholinesterase and digestive enzymes (trypsin and amylase). A concentration dependant increase in the activities of antioxidant enzymes such as catalase, superoxide dismutase and glutathione S-transferase suggested the generation of oxidative stress in M. macrocopa after AuNPs exposure. Changes in enzyme activity can be utilized as biomarker(s) for early detection of nanoparticle contamination in aquatic habitat. AuNPs accumulation in gut of M. macrocopa increased the metal bio burden (11 mg/L) and exhibited inhibitory action on digestive enzymes. Complete depuration of AuNPs was not observed after transferring nano-exposed M. macrocopa to normal medium without AuNPs. AuNPs tended to adhere on external body parts such as setae, carapace of M. macrocopa which interfered with swimming activity and also changed the behavioral pattern. AuNPs underwent agglomeration in the medium used for maintenance of M. macrocopa. As nanomaterials are emerging pollutants in aquatic systems, the present work highlights the hazardous effect of AuNPs and development of enzymatic biomarkers to curtail it at community level.