The aim of the study described here was to prospectively investigate the clinical and practical value of percutaneous contrast-enhanced ultrasound (CEUS)-guided biopsy of pancreatic lesions that are not definitively localized by B-mode ultrasound (US). Fifty-three patients underwent CEUS-guided biopsy. The rate of satisfactory percutaneous biopsy was 96.23% (51/53) with a median number of puncture attempts per patient of 3.0, and the diagnostic accuracy in satisfactory sampling was 96.08% (49/51). The sensitivity of CEUS-guided biopsy in diagnosing malignancy was 90.48% (38/42). There was only one major complication in our study, a patient (1/51, 1.96%) with biliary peritonitis. For pancreatic lesions that are not definitively localized by B-mode US, puncture guided by CEUS could improve accuracy, lower the incidence of complications and avoid unnecessary biopsy.