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In vivo optical Imaging is an inexpensive and highly sensitive modality to investigate and follow up diseases like breast cancer. However, fluorescence labels and specific tracers are still works in progress to bring this promising modality into the clinical day-to-day use. In this study an anti-MUC-1 binding single-chain antibody fragment was screened, produced and afterwards labeled with newly designed and surface modified NaYF4:Yb,Er upconversion nanoparticles as fluorescence reporter constructs. The MUC-1 binding of the conjugate was examined in vitro and in vivo using modified state-of-the-art small animal Imaging equipment. Binding of the newly generated upconversion nanoparticle based probe to MUC-1 positive cells was clearly shown via laser scanning microscopy and in an initial proof of principal small animal optical imaging approach.