Toxocara canis is one of the most common intestinal parasites in dogs and represents a highly infectious zoonotic parasite worldwide. Adult worms live in the bowel of dogs, and infections in puppies are commonly acquired transplacentally. The biology of the parasite and the commonly used diagnostic method, based on faecal examination, often prevent an early diagnosis of toxocariasis in puppies. Ultrasonographic diagnosis of intestinal ascariasis could be a feasible alternative method to diagnose T. canis infection in puppies during the prepatent period. The present study evaluated the diagnostic accuracy of ultrasonography for the diagnosis of intestinal toxocariasis in new-born puppies during the prepatent period. Fifty-four new-born puppies were prospectively recruited in the study. Each dog underwent intestinal ultrasonography and copro-microscopic examination every 5 days, starting from the 10th day post-birth. Intestinal ultrasonography visualized adult T. canis nematodes in the puppies' small intestine from the 10th day post-birth. On the 15th day after birth, ultrasonography showed 100% specificity and 85.4% sensitivity in diagnosing T. canis infection, despite negative results deriving from the copro-microscopic examination performed at the same time point. Our results showed that ultrasonography can be used as test for early diagnosis of T. canis infection in new-born puppies during the prepatent period. Early ultrasonographic diagnosis of T. canis infection in puppies could help control the disease in dogs and reduce the zoonotic risk for the human population.