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Biosynthesis methods employing microorganisms have emerged as an eco‐friendly, clean, and viable alternative to chemical and physical processes. The present study reports the synthesis of copper oxide nanoparticles (CuONPs) using cell‐free culture supernatant of marine Streptomyces sp. MHM38. For the optimized production of CuONPs, the influence of some parameters, such as the concentration of copper sulfate (CuSO₄), reaction time, filtrate to substrate ratio, and pH, was studied. 5 mM of CuSO₄ was optimal for nanoparticle (NP) production. Well‐defined CuONP formation occurred after 60 min of incubation when an equal volume of filtrate (cell‐free supernatant) to substrate (CuSO₄ solution) was added. UV‐visible spectroscopy analysis of CuONPs exhibited a peak at 550 nm, which corresponds to the surface plasmon resonance of CuONPs. Most of the particles were spherical and were 1.72–13 …