Background: Antibody-drug conjugates such as Trastuzumab deruxtecan significantly improved progression-free and overall survival for the HER2 low expression (HER2-low) breast cancer (BC) patients, pushing the research field of HER2-low to a new climax. In fact, the traditional immunohistochemistry (IHC) to identify the HER2-low or HER2-negative expression (HER2-neg) has been challenged, and it cannot predict the prognosis of HER2-low/neg patients well. Methods: Three pathologists evaluated the HER2 expression status of the subjects via IHC. More than two pathologists judged HER2 0-2 and FISH negative samples were included in this trial. ERBB2 mRNA level of formalin-fixed, paraffin-embedded BC post-surgery specimens were detected by MammaTyper quantitative real-time polymerase chain reaction (qRT-PCR) kit. The consistency of HER2 immunohistochemistry (IHC) and MammaTyper results were analyzed by interclass correlation coefficients (ICC). Differences in disease-free survival (DFS) were assessed by Kaplan Meier analysis. Results: 793 cases were included. The ICC value between IHC and MammaTyper, assessed for HER2-neg vs HER2-low is 0.28. With a median follow up of 2.5 years, there were 36 events in the whole subjects. In the whole subjects, MammaTyper successfully showed that the prognosis of HER2-low was significantly better than HER2-neg (P < 0.001, HR = 3.497, 95%CI,1.807-6.77), but IHC could not distinguished them (P = 0.1071, HR = 1.776, 95%CI, 0.9194-3.43). Although the follow-up time is short, HER2-low subjects also showed better prognosis than HER2-neg in TNBC subjects distinguished by MammaTyper (P = 0.0271, HR = 2.912, 95%CI,1.129-7.511), not by IHC (P = 0.1032, HR = 2.213, 95%CI, 0.8512-5.756). However, in 291 cases of IHC and MammaTyper detection discordancy group, IHC results showed that the prognosis of HER2-low was worse, which was contrary to the MammaTyper results and the results of the above overall test results, suggesting that IHC could not verily reflect the amount of HER2 expression in some samples, and the prognosis of HER2-low population might be more related to the mRNA expression of HER2. Conclusions: Compared with IHC/FISH, MammaTyper qRT-PCR assay may actually reflect the prognosis of HER2-neg/low, which is expected to become a diagnostic beacon for HER2-low target drugs.