Fluorescent nanoparticles are promising tools for living cancer cell imaging and cancer targeting. In this study, estrogen conjugated dye-doped fluorescent nanoparticles (estrogen conjugated FNPs) were synthesized and characterized. The functionalized nanoparticles with low toxicity have shown high selectivity and sensitivity toward target cells. Based on the specific recognition between the estrogen and the estrogen receptor, estrogen conjugated FNPs have been employed as optical probes for specific targeting of estrogen receptor-positive cancer cells with fluorescence microscopy imaging technology. The results demonstrate that the estrogen conjugated FNPs can effectively recognize breast cancer cells with good sensitivity and exceptional photostability, which would offer a novel approach for the diagnosis of breast cancer cells, as well as a new method in detecting estrogen receptors.