We aimed to evaluate the value of sentinel lymph node contrast-enhanced ultrasound (SLN-CEUS) and surface tracing for the biopsy of intra-operative sentinel lymph nodes (SLNs). Between June 2015 and December 2017, a total of 453 patients with early invasive breast cancer were recruited. Patients received an intradermal injection of microbubble contrast agent around the areola on the day before surgery. The locations and sizes of lymphatic channels (LCs) and SLNs were marked on the body surface using gentian violet. Then, injection of double blue dye was performed half an hour before surgery. We compared the pathway of LCs and the location of SLNs obtained from SLN-CEUS and blue dye during surgery. Among the 453 patients, the mean numbers of LCs and SLNs detected by SLN-CEUS were 1.42 and 1.72, respectively, and the coincidence rate was 98.2% compared with blue dye during surgery. The median distance from the SLN to skin measured by preoperative CEUS and blue dye was 1.95 § 0.69 and 2.03 § 0.87 cm (p = 0.35). There were three SLN enhancement in our research, including homogeneous enhancement, inhomogeneous enhancement and no enhancement, with the sensitivity, specificity, positive predictive value and negative predictive value of SLN-CEUS for the diagnosis of SLNs being 96.82%, 91.91%, 87.54% and 98.01%, respectively. SLN-CEUS with skin marking can identify the pathway of LCs and the location of the SLN before surgery, measure the distance from the SLN to skin and determine if the SLN is metastatic. SLN-CEUS can be used as an effective complement to the blue dye method.