Proteins belonging to the linear ubiquitin assembly complex (LUBAC) are believed to be important in tumorigenesis. LUBAC has been demonstrated to be composed of RBCK1, RNF31 and SHARPIN. The aim of this study was to explore all members of the LUBAC complex as novel biomarkers in breast cancer. We have already reported that RNF31 mRNA levels are higher in breast cancer samples compared to adjacent non-tumor tissue. In this study we extend these findings by demonstrating that the mRNA levels of RBCK1 and SHARPIN are also higher in tumors compared to adjacent non-tumor tissue in the same cross sectional study of samples (p < 0.001). In addition, up-regulated mRNA expression of all three members of the LUBAC complex displayed high predictive value in distinguishing tumor tissues from adjacent non-tumor tissue as determined by ROC curve analysis. Furthermore, we investigated whether there is an association between the mRNA and protein expression levels of RBCK1, RNF31 and SHARPIN and clinicopathological parameters including estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2) status and found that RNF31 protein is significantly higher in ERalpha-negative tumors than ERalpha-positive tumors (p = 0.034). Collectively, our findings indicate that up-regulated mRNA expression of RNF31, RBCK1 and SHARPIN could potentially be diagnostic biomarkers of breast cancer and RNF31 might be a drug target for ERalpha-negative breast cancers.