Purpose To predict human epidermal growth factor receptor 2 (HER2) expression in breast cancer (BC) using Sonazoid-enhanced ultrasound in a machine learning-based model.Materials and methods Between August 2020 and February 2021, patients with breast cancer who underwent surgical treatment without neoadjuvant chemotherapy were prospectively enrolled from 17 hospitals in China. HER2 expression status was assessed by immunohistochemistry or fluorescence in situ hybridization (FISH). The training set contained data from 11 hospitals and the validation set contained 6 hospitals. Clinical features, B-mode ultrasound, contrast-enhanced ultrasound (CEUS), and time-intensity curve were selected by the Least Absolute Shrinkage and Selection Operator. Based on the selected features, six prediction models were established to predict HER2 3 + and 2 +/1 + expression: logistic regression (LR), support vector machine (SVM), random forest (RF), eXtreme Gradient Boosting (XGB), XGB combined with LR, and fusion model.Results A total of 140 patients with breast cancer were enrolled in this study. Seven features related to HER2 3 + and six features related to HER2 2+/1 + were selected to establish prediction models. Among the six models, LR, SVM, and XGB showed the best prediction performance for both HER2 3 + and HER2 2+/1 + cases. These three models were then combined into a fusion model. In the validation, the fusion model achieved the highest value of area under the receiver operating characteristic curve as 0.869 (95%CI: 0.715-0.958) for predicting HER2 3 + and 0.747 (95%CI: 0.548-0.891) for predicting HER2 2+/1 + cases. The model could correctly upgrade HER2 2 + cases to HER2 3 + cases, consistent with the FISH test results.Conclusion Sonazoid-enhanced ultrasound can provide effective guidance for targeted therapy of breast cancer by predicting HER2 expression using machine learning approaches.