The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that is expressed in most normal human epithelia and overexpressed in most carcinomas. This molecule is responsible for cell-to-cell adhesion and additionally participates in signalling, cell migration, proliferation and differentiation. Therefore, EpCAM has been the target of immunotherapy in clinical trials of several solid tumours. It has been found to play an important role in the detection and isolation of circulating tumour cells (CTCs). The aim of this study was to investigate and compare the specificity and selectivity of different anti-EpCAM antibodies in order to assess their usefulness for CTCs binding. All experiments were performed in six different types of breast cancer cell lines (MCF-7, SkBr-3, T47D, CAMA-1, MDAMB-231, and BT-20) and using three different anti-EpCAM antibodies (EBA-1, AUA-1, and 9C4). Immunofluorescence and Real-Time PCR techniques were applied to analyse the protein and gene expression levels. The experiments revealed that the investigated antibodies differed significantly regarding the specificity of EpCAM antigen binding. The most significant role in targeting CTCs was played by the EBA-1 and 9C4 anti-EpCAM antibodies. They revealed the strongest immunofluorescent signal among other applied antibodies and/or were specific for all examined breast cancer cell lines. The strength and specificity of reaction was dependent not only on the type of antibody, but also on the type of breast cancer cell line. We noted that the diverse sensitivities of reactions depended on the type of applied antibody. We therefore recommend the simultaneous application of different anti-EpCAM antibodies. An appropriate choice of anti-EpCAM antibodies and an evaluation of EpCAM expression in breast cancer appear to be crucial, especially as this antigen is being proposed as a marker for the detection of circulating tumour cells.