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This study aimed to assess the application of ultrasonography for preoperative diagnosis of retroperitoneal fibrosis (RPF). A total of 51 patients with clinically suspected RPF underwent transabdominal ultrasonography and ultrasound-guided histopathologic biopsy (tAU-UGHB). Retroperitoneal fibrosis was diagnosed in 35 patients; of these, 31 cases (88.6%) received a diagnosis using tAU-UGHB. The ultrasonographic images mainly showed solid, irregular isoechoic masses, which were present behind the retroperitoneum and surrounded the abdominal aorta, inferior vena cava, and ureters and had clear borders with the encapsulated structures. Compared with other retroperitoneal lesions, RPF lesions were mainly located below the initial level of the renal artery, and their internal flow signals were not rich (P < 0.05). Retroperitoneal fibrosis had characteristic ultrasonographic features, and color Doppler ultrasound-guided biopsy could be positioned accurately, safely, and efficiently; therefore, tAU-UGHB can be used as an effective modality for preoperative diagnosis of RPF.