Targeted therapy is essential for the 15-30% invasive breast cancer patients with human epidermal growth factor receptor 2 (HER2) over-expression. However, current HER2 diagnosing methods rely on complex manual works and highly subjective interpretations. To more accurately and objectively assess the HER2 amplification status of formalin fixed paraffin embedded (FFPE) samples, a droplet digital PCR (ddPCR) assay based on multi-reference genes was developed. We established a four-fluorescence ddPCR assay using breast cancer cell lines (T-47D and SK-BR-3) and validated it on 101 clinical breast cancer FFPE samples. Compared to clinicopathological results, the ddPCR assay based on two out of three reference genes demonstrated superior sensitivity (82.6%), specificity (98.7%), and consistency (95.0%) in determining HER2 status over assays using single or three reference genes. Whole genome sequencing of the abnormal cases further confirmed that the ddPCR assay outperformed clinical immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and quantitative PCR (qPCR) in accuracy. Our findings demonstrate that the multi-reference gene ddPCR assay significantly improves the accuracy of HER2 status detection and reduces errors associated with chromosome 17 abnormalities. This method holds promise as a complementary or alternative approach to conventional IHC and FISH testing in tissue biopsies and is also feasible for liquid biopsies.