This study aims to evaluate the performance of contrast-enhanced ultrasound (CEUS) and biological tests to characterize portal vein thrombosis (PVT). We retrospectively analyzed 101 patients with PVT, liver cirrhosis, and hepatocellular carcinoma (HCC). In all patients, demographic, biologic, imaging, and endoscopic data were collected. All patients underwent CEUS and a second line imaging technique (CE-CT/MRI) to characterize PVT. Of the 101 cirrhotic subjects, 77 (76.2%) had HCC. CEUS had 98.6% sensitivity (Se) and 89.3% specificity (Sp) for the characterization of PVT type. A significant correlation was found between alpha-fetoprotein (AFP) levels and the PVT characterization at CEUS (r = 0.28, p = 0.0098) and CT/MRI (r = 0.3, p = 0.0057). Using the AFP rule-out cutoff values for HCC (AFP p = 0.025) or AFP serum levels (AUC—0.99 vs. AUC—0.96, p = 0.047) for characterizing PVT. In conclusion, CEUS is a sensitive method for the characterization of PVT. The PVT score had the highest performance for PVT characterization.