Zn1−xCoxO thin films with c-axis preferred orientation were deposited on sapphire (0001) by pulsed laser deposition (PLD) technique at different substrate temperatures in an oxygen-deficient ambient. The effect of substrate temperature on the microstructure, morphology and the optical properties of the Zn1−xCoxO thin films was studied by means of X-ray diffraction (XRD), atomic force microscopy (AFM), UV–visible–NIR spectrophotometer, fluorescence spectrophotometer. The results showed that the crystallization of the films was promoted as substrate temperature rose. The structure of the samples was not distorted by the Co incorporating into ZnO lattice. The surface roughness of all samples decreased as substrate temperature increased. The Co concentration in the film was higher than in the target. Emission peak near band edge emission of ZnO from the PL spectra of the all samples was quenched because the dopant complexes acted as non-radiative centers. While three emission bands located at 409nm (3.03eV), 496nm (2.5eV) and 513nm (2.4eV) were, respectively, observed from the PL spectra of the four samples. The three emission bands were in relation to Zn interstitials, Zn vacancies and the complex of VO and Zni (VOZni). The quantity of the Zn interstitials maintained invariable basically, while the quantity of the VOZni slightly decreased as substrate temperature increased.