One environmentally friendly technique for creating colloids of nanoparticles is pulsed laser ablation in liquid, which stands out for its ability to create nanoparticles free of contamination without the use of hazardous chemicals. In the present work, A Q-Switched Nanosecond Nd: YAG laser was utilized to precisely produce colloidal copper oxide nanoparticles (CuONPs) through the laser ablation of a copper target immersed in distilled water. The impact of the laser fluence and laser ablation time on the size, morphology, and concentration of the generated CuONPs was investigated experimentally. The UV–visible absorption spectra of CuONPs display surface plasmonic resonance peaks in the UV region, which are attributed to interband transitions. The energy band gaps of the colloidal CuONPs were determined using UV–visible spectroscopy, and they ranged between 3.45 and 3.72 eV at various ablation times and laser fluences. The TEM micrographs were used to assess the size and morphology of the CuONPs, while the concentration and elemental composition were determined using ICP and EDXA spectroscopy. The TEM images of the CuONPs indicate a spherical shape at various laser ablation times and laser fluences. The average size of the CuONPs decreased to the quantum size range. The experimental results show that increasing laser fluence from 28.6 to 51.5 × 103 J/cm2 while keeping laser ablation time fixed for 30 min resulted in a reduction in the average size of the nanoparticles from 8.5 to 4.6 nm, respectively. Moreover, increasing laser ablation time from 10 to 50 min at a constant incident laser fluence of 34.3 × 103 J/cm2 decreased the average size of the CuONPs from 6.8 to 4.3 nm, respectively. The concentration of the synthesized CuONPs increased as laser fluence and laser ablation time increased.