In this work, the effect of substrate temperature (ST) during deposition and dopant amount of Mn (1,3,5, and 7 %) and Y(3 %), (Mn/Y), on the physical properties of copper oxide (CuO) the nanostructured film were analyzed using different diagnostic tools. The pure and (Mn, Y) co-doped CuO thin films were deposited by the spray pyrolysis method, which is effective and easy to apply for any substrate. Deposited pure and (Mn,Y) co-doped CuO nanostructured films were investigated by X-ray diffraction (XRD), UV–Vis spectroscopy, and Field emission scanning electron microscopy (FESEM) techniques to analyze the influence of ST and co-doping on the structural, morphological, and optical properties of prepared films. The FESEM is used to visualize nanostructures on the surface of the film. Also, it is observed that the surface morphology of CuO film changes from a spherical-like to a nanocloumnar-like structure with increasing substrate temperature. The XRD diffractograms confirm the monoclinic crystal structure of CuO, in polycrystalline nature for all deposited films. UV–Vis spectra suggest changing the optical absorbance and the band gap of the pure and Mn/Y co-doped CuO nanostructured films. In the first investigation of this study, the better substrate temperature was obtained as 400 °C according to results. In the second part, the effect of Y and Mn doping with different content was analyzed for the CuO film deposited at the chosen ST of 400 °C. There is no observable crystalline structure variation in the XRD pattern, but the size of nanostructure and dislocation density are varied with Mn/Y co-doping. FESEM micrographs confirm the impact of the co-doping concentration on the size, shape, and surface properties of CuO nanostructured film. In addition, the optical band gap increases with Mn doping up to 3 % of Mn content and it was determined as 2.69 eV for the sample 3Mn/Y@CuO nanostructured film. The obtained results suggest that the substrate temperature has an important impact on the physical properties of the spray deposited CuO nanostructured film, and the structural, optical, and surface properties of the CuO nanostructured films may be engineered by Mn/Y doping concentration.