Antibiotic contamination has become a global environmental issue of widespread concern, among which oxytetracycline contamination is very severe. In this study, earthworm (Eisenia fetida) was exposed to oxytetracycline to study its impact on the soil environment. The total protein (TP), catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), glutathione S-transferase (GST), and glutathione peroxidase (GPX) oxidative stress indicators in earthworms were measured, and the integrated biomarker response (IBR) approach was used to evaluate the toxic effect of oxytetracycline on earthworms. A Technique for Order Preference by Similarity to an Ideal Solution (TOPSIS) and a path analysis model were used to explore the physiological and metabolic processes of earthworms after stress occurs. The results showed that SOD, GPX, and GST play important roles in resisting oxytetracycline stress. In addition, stress injury showed a good dose–effect relationship, and long-term stress from pollutants resulted in the most serious damage to the head tissue of earthworms. These results provide a theoretical basis for understanding the toxic effect of oxytetracycline on soil animals, monitoring the pollution status of oxytetracycline in soil, and conducting ecological security risk assessment.