Di-n-butyl phthalate (DBP), a widely used plasticizer in the plastic industry, affects regulation of the endocrine system and causes toxicity in animals. In the present study, we evaluated a series of ecotoxicological stress biomarkers in the common carp (Cyprinus carpio) as an experimental model to test for alterations in gene expression at a sublethal concentration of 1 mg/L DBP for 4, 24, and 96 h. In gills, an immediate increase in CYP1A messenger RNA (mRNA) levels was observed within the first 4 h and persisted for 96 h. Protein levels were nearly consistent with mRNA levels. However, a time-dependent inhibition was observed in CYP1A levels in the liver within 96 h. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels increased gradually in liver with exposure time to a maximum level of 11-fold. Varied responses of different tissues were likely due to xenobiotic metabolism of DBP. In conclusion, evaluating the tissue-specific alterations of CYP1A, SOD, and GPx levels can be used as specific and effective biomarkers for ecotoxicological monitoring of DBP pollution. We strongly recommend using molecular tools to ecotoxicologists for aquatic monitoring of newly emerging pollutants.