Novel 17 alpha-Etinylestradiol-substituted 4,4-difluoro-4-borata-3a, 4a-diaza-s-indacene (BODIPY) dyes were prepared as fluorescent imaging probes. The identities of newly synthesized dyes were fully characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and H-1 and C-13 nuclear magnetic resonance (NMR) spectroscopy. The photophysical properties (molar extinction coefficient, fluorescence lifetime and fluorescence quantum yield) of the compounds were investigated by means of absorption and fluorescence spectroscopies in dilute ethanol solutions. We were also interested in the live cell imaging studies of the 17 alpha-etinylestradiol-linked BODIPYs, by particularly concerning their ability to monitoring breast cancer cell lines. These studies showed that one of the novel 17 alpha-etinylestradiol-substituted BODIPY dyes was able to distinguish breast cancer subtypes. This is the first study sub-categorizing breast cancer via fluorescent-tagged estrogen analog, which is critical in terms of improving the molecular diagnostics and eliminating the drawbacks of available methods. These studies are very selective up to date for a BODIPY-based fluorescent label and define a pivotal biological importance of BODIPY as a building block in live cell imaging studies.