Here we propose a simple and reliable approach for detection of the tumor marker HER2/neu using the targeting fluorescent hybrid protein DARPin-mCherry. As a targeting module, we used DARPin9-29, which is a member of a novel class of non-immunoglobulin targeting proteins that can highly selectively recognize the extracellular domain of the epidermal growth factor receptor HER2/neu. The red fluorescent protein mCherry was used as the detecting module. The hybrid protein DARPin-mCherry was prepared with high yield in a bacterial expression system and purified in one step by affinity chromatography. The purified protein is not prone to aggregation. The specificity of DARPin-mCherry binding with the HER2/neu tumor marker was demonstrated using confocal microscopy, flow cytofluorimetry, and surface plasmon resonance. The dissociation constant of the DARPin-mCherry protein complex with the HER2/neu receptor determined by surface plasmon resonance was calculated to be 4.5 nM. These characteristics of the hybrid protein DARPin-mCherry suggest it as a promising agent for immunofluorescent assay and an attractive alternative to antibodies and their fragments labeled with fluorescent dyes that are now used for this purpose.