This study aimed to evaluate the usefulness of sentinel lymph node (SLN) detection in breast cancer using contrast-enhanced ultrasonography (CEUS) with subareolar Sonazoid injection. The subjects were 20 breast cancer patients. General anesthesia was induced and 2 mL of Sonazoid was injected subareolarly. After massage of the injection site, the axillary area was observed transdermally using coded phase inversion harmonic ultrasonography with mechanical indices of 0.15 to 0.19. When contrast-enhanced lymph nodes (LNs) were seen, they were defined as CE-SLN. Two other SLN detection methods, the gamma-probe-guided and dye-guided methods, were performed together. We evaluated the SLNs detected by each method to determine if they corresponded with each other and calculated the SLN detection rate. After the SLNs were resected, pathologic examinations were done. The SLN detection rate of the CEUS-guided method, the dye-guided method and the gamma-probe-guided method were 70%, 75% and 100%, respectively. There was no statistically significant difference in these rates between the CEUS-guided and dye-guided methods (p = 0.99) but the CEUS-guided method showed a significantly lower rate than the gamma-probe-guided method (p = 0.020), and dye-guided method also showed a significantly lower rate than the gamma-probe-guided method (p = 0.047). The number of CE-SLNs was 1 or 2 (average 1.1) and each took 2 to 20 (average 5.3) min to detect. The CE-SLNs corresponded grossly with SLNs detected by the gamma-probe-guided and dye-guided methods. The pathologic results indicated no metastasis from the resected SLNs in 15 of 20 cases. However, the CEUS-guided method detected 12 cases of these 15 and CE-SLNs were detected in two of the remaining five metastasis cases. In summary, in breast cancer patients, after subareolar injection of Sonazoid, contrast-enhanced LNs were observed in real time with ultrasonography. In an initial clinical study of 20 cases, the detection rate of the CEUS-guided method was less than that of the gamma-probe-guided method. It is suggested that the CEUS-guided method using Sonazoid may, with some improvements, be a useful new modality for sentinel node identification.