In the present study, selenium nanoparticles (SeNPs) were prepared via selenious acid reduction with ascorbic acid in aqueous media and used for immobilization of hydrolytic enzyme chymotrypsin (CT). It was shown that CT is an effective stabilizer of SeNPs in solution at acidic pH values. CT did not stabilize SeNPs upon reaction in an alkaline range when 2-mercaptoethanol was used as reducing agent. UV- and visible spectroscopy and dynamic light scattering showed that synthesis temperature and concentration ratio of components play important role in the formation of Se-CT nanocomplexes. An increase in temperature of synthesis shifted the maximum of optical density toward longer wavelengths: 256, 270, and 285 nm for nanocomplexes obtained at temperature of 4, 20, and 37°C, respectively (for all studied components concentration ratios). Hydrodynamic radii of nanocomplexes also changed depending on the synthesis temperature and components concentration ratio. Aggregative stability of nanocomplexes with equal concentrations of Se and CT was studied in a wide range of media pH. Nanocomplexes aggregatively stable in solution were observed in the acidic pH range (2.8–3.75) and the alkaline range (7.1–10.5). The pH values at which the full phase separation in the system was observed lay in the range of 3.75–4.0 to 6.5–7.0. Increasing the concentration of protein stabilizer extended the pH intervals of the aggregatively stable state of nanocomplexes. Immobilization of CT on SeNPs do not lead to significant decrease of the enzyme activity.