A novel approach using a zwitterionic sulfobetaine-based surfactant for the synthesis of spherical copper oxide nanoparticles (Cu₂O NPs) has been applied. For the first time, N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate has been used as stabilizer to control the size and morphology of Cu₂O NPs. Several techniques, such as transmission electron microscopy (TEM), X-ray diffraction (XRD), and fluorescence spectroscopy, are used to investigate the size, structure, and optical properties of synthesized Cu₂O nanocrystals. The results indicate that copper(I) oxide nanoparticles with size in the range of 2 to 45 nm and crystalline structure, exhibit intense yellow fluorescence (λ_em = 575 nm). Furthermore, the cytotoxicity studies show that sulfobetaine-stabilized copper oxide nanoparticles prompt inhibition of cancer cell proliferation in a concentration-dependent manner, however, the adverse effect on the normal cells has also been observed. The results indicate that the sulfobetaine-stabilized Cu₂O, because of their unique properties, have a potential to be applied in medical fields, such as cancer therapy and bioimaging.