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Physiological and endocrine maintenance of a normal human growth hormone (hGH) concentration is crucial for growth, development, and a number of essential biological processes. In this study, we describe the preparation and characterization of magnetic nanoparticles coated with a gold shell (MNPs-Au). The optimal surface concentration of monoclonal anti-hGH antibodies (m-anti-hGH) on magnetic nanoparticles, as well as conditions that decrease non-specific interactions during the magneto-immunoassay, were elaborated. After the selective recognition, separation, and pre-concentration of hGH by MNPs-Au/m-anti-hGH and the hGH interaction with specific polyclonal biotin-labeled antibodies (p-anti-hHG-B) and streptavidin modified horseradish peroxidase (S-HRP), the MNPs-Au/m-anti-hGH/hGH/p-anti-hGH-B/S-HRP immunoconjugate was formed. The concentration of hGH was determined after the addition of 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide substrate solution for HRP; the absorbance at 450 nm was registered after the addition of STOP solution. The developed sandwich-type colorimetric magneto-immunoassay is characterized by a clinically relevant linear range (from 0.1 to 5.0 nmol L⁻¹, <i>R</i>² 0.9831), low limit of detection (0.082 nmol L⁻¹), and negligible non-specific binding of other antibodies or S-HRP. The obtained results demonstrate the applicability of the developed magneto-immunoassay for the concentration and determination of hGH in the serum. Additionally, important technical solutions for the development of the sandwich-type colorimetric magneto-immunoassay are discussed.