The aim of the investigation was to create multifunctional antitumor protein agent DARPin-mCherry-PE40 possessing both diagnostic and therapeutic properties. Materials and Methods. The experiments were carried out on human cell lines of breast adenocarcinoma SK-BR-3. Chinese hamster ovarian cell line CHO-K1 was used as a control. Genetic engineering methods, metal chelate affinity chromatography, confocal microscopy and spectrophotometric MTT test were used to perform the experiments. Results. A multifunctional targeted anticancer protein agent DARPin-mCherry-PE40 consisting of targeted HER2-specific nonimmunoglobulin module DARPin, an imaging module mCherry and cytotoxic module based on a fragment of Pseudomonas exotoxin A (PE40) has been created. Using HER2-positive cells it was shown in vitro that all three domains of the recombinant protein retained their functional qualities — high affinity for HER2 antigen, ability to fluorescence, and cytotoxicity. Conclusion. The ability of the targeted antitumor agent DARPin-mCherry-PE40 selectively stains HER2-positive cells and highly selective cytotoxicity against these cells make this targeted recombinant protein DARPin-mCherry-PE40 a perspective theranostic agent for the diagnosis and treatment of HER2-positive human tumors.