Surface modification of nanoparticles with various biologically active molecules makes it possible to realize the huge biomedical potential of various nanoobjects. Functionally active particles are usually obtained through stabilization by polymers and subsequent chemical conjugation with biomolecules, such as, antibodies or aptamers. This entails a number of problems, such as non-oriented conjugation, low coupling yield and the inability to easily vary the attached components on demand. To solve this problem, we developed a novel method of magnetite nanoparticles stabilization with simultaneous modification by functionally active protein – Barstar, for subsequent self-assembly of the necessary components with these particles through the interaction of high-affinity protein pair Barnase∗Barstar (Kaff= 1014M−1). Namely, we developed a biocompatible Bs-C-Mms6 fusion protein containing the C-terminal part of the Mms6 (magnetite-binding protein of magnetotactic bacteria) and Barstar (an inhibitor of bacterial ribonuclease Barnase). We obtained stable in PBS magnetite nanoparticles modified with Bs-C-Mms6. These particles can be used for self-assembly with any type of Barnase-containing molecules. To demonstrate the effectiveness of this approach for the development of targeted nanoparticles, we performed a self-assembly of these particles with a fusion protein of Barnase and DARPin9.29, namely DARPin9.29-Bn. DARPin9.29 recognizes the extracellular domain of clinically important HER2/neu oncomarker. We have shown that such particles selectively bind this oncomarker and can be used to detect HER2/neu-positive cancer cells for diagnostic purposes.