In this study, the sublethal effects of caffeine, sulfamethoxazole and their mixture on goldfish (Carassius auratus) were investigated, the biomarkers including acetylcholinesterase (AChE) in brain, 7-ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST) and superoxide dismutase (SOD) in liver and vitellogenin (VTG) in serum were determined after 1, 2, 4, and 7 days of exposure. AChE activity was significantly inhibited by caffeine (≥0.4 mg/l), sulfamethoxazole (≥0.4 mg/l) and their mixtures (≥0.048 mg/l) during all exposure periods, and obvious concentration-response and time-response relationships were obtained. EROD, GST and SOD activities were significantly increased by individual compounds and mixtures in most cases. GST induction exhibited bell-shaped concentration-response curves. Serum VTG was significantly induced by 2 mg/l of caffeine, 10 mg/l of sulfamethoxazole and the mixtures at concentrations ≥1.2 mg/l. In general, the two pharmaceuticals induced similar biological responses. The joint effect of caffeine/sulfamethoxazole was additive with regard to AChE and GST activity variation and was antagonistic with regard to EROD and SOD induction. The results indicated that multiple biomarker response method might be a useful tool for describing an integrated toxicological effect of chemicals. VTG induction suggested that caffeine and sulfamethoxazole may cause a slightly feminization effect.