The rapid determination of metastasis in sentinel lymph nodes (SLNs) of breast cancer patients plays a significant role in the selection of a surgery strategy. Although a previous one-step nucleic acid amplification assay that uses reverse-transcription (RT) loop-mediated isothermal amplification (LAMP) has showed specific advantages over traditional pathological examination, its target marker requires optimisation. In addition to epithelial-specific CK19, the internal control gene PBGD and the breast-specific PIP were included in the new method. After the RT-LAMP primers were designed and verified using a cell line, the performance of our method was evaluated by comparing it with the corresponding result of the Food and Drug Administration approved breast lymph node (BLN) assay and routine pathological examination. One hundred and seventy-four valid SLN samples from 101 patients were collected from five hospitals. The threshold of reaction time for CK19, PIP and PBGD was defined as 16, 20 and 20 min, respectively. Compared with the BLN assay, the concordance rate of our method was 95.4 % (166/174). Statistical analysis revealed that the two methods are consistent (kappa = 0.890, P < 0.001). When compared with pathological examination, the performance of our method (sensitivity = 81.3 %, specificity = 89.7 %, kappa = 0.691, P < 0.001) was similar to that of the BLN assay (sensitivity = 87.5 %, specificity = 84.9 %, kappa = 0.668, P < 0.001). This result demonstrates the potential usefulness of our method in clinical practice. In conclusion, we preliminarily established an intra-operative diagnostic method that assimilates the merits of previous assays. In contrast with the BLN assay and pathological examination, our method can be completed in 30 min and shows high sensitivity, specificity and consistency, which we consider as promising for clinical application.