This study investigated the effect of an ammonia nitrogen-reducing biofilm on the stress response and muscle quality of crucian carp (Carassius auratus) during high-density transportation. A control group and a treatment group (with ammonia nitrogen-reducing biofilm) were established, and multiple physiological and quality indicators were measured under simulated transportation conditions. The results showed that the biofilm significantly alleviated transportation stress, as evidenced by smaller increases in blood stress indicators (e.g., cortisol and glucose). Meanwhile, it reduced gill and liver damage, increased antioxidant enzyme activity, and decreased malondialdehyde (MDA) content. In terms of muscle quality, the treatment group exhibited lower drip loss and lactic acid content, higher shear force, pH, glycogen, and ATP levels, less color change, reduced cell apoptosis, more intact muscle tissue structure, and significantly improved survival rates. This study provides an effective technical reference for the high-density live transportation of freshwater fish.