Noninvasive early detection of breast cancer through the use of biomarkers is urgently needed since the risk of recurrence, morbidity, and mortality is closely related to disease stage at the time of primary surgery. A crucial issue in this approach is the availability of relevant markers and corresponding monoclonal antibodies suitable for the development of effective immunodiagnostic modalities. The identification of such markers from human pathological lesions and the isolation of specific antibodies using conventional approaches remain major challenges. Camelids produce functional antibodies devoid of light chains in which the single N-terminal domain of the heavy chain is fully capable of antigen binding. When produced as an independent domain, these so-called single-domain antibody fragments (sdAbs) or nanobodies have several advantages for biotechnological applications owing to their unique properties of size (13 kDa), stability, solubility, and expression yield. In this work, we have generated phage display libraries from animals immunized with breast cancer biopsies. These libraries were used to isolate sdAbs against known and relevant antigens such as HER2, or several cancer-specific sdAbs against unknown targets. We describe the identification of one these targets, cytokeratin 19, using affinity purification in combination with mass spectrometry. Some of these sdAbs were used in several straightforward diagnostic applications such as immunohistochemical analysis of tumor samples, multiplexed cytometric bead array analysis of crude samples, or an immune enrichment procedure of rare cells. Here, we demonstrate that phage display-based selection of single-domain antibodies is an efficient and high-throughput compatible approach to generate binders with excellent characteristics for the fast development of diagnostic and prognostic modalities.