The aim of this study was to investigate the optimization of spatial resolution and image reconstruction parameters related to image quality in an iterative reconstruction algorithm for the small-animal MetisTM PET/CT system. We used a homemade Derenzo phantom to evaluate the image quality by visual assessment, signal-to-noise ratio, contrast, coefficient of variation, and contrast-to-noise ratio of the 0.8 mm hot rods of 8 slices in the centre of the phantom PET images. A healthy mouse study was performed to analyze the influence of optimal reconstruction parameters and Gaussian post-filter FWHM. In the phantom study, the best image quality was obtained by placing the phantom at one end, keeping the central axis parallel to X-axis of the system, selecting iterations between 30 and 40, with a reconstruction voxel of 0.314 mm and a Gaussian post-filter FWHM of 1.57 mm. The optimization of spatial resolution can reach 0.6-mm. In the animal study, it was suitable to choose a voxel size of 0.472-mm, iterations between 30 and 40, and 2.36-mm Gaussian post-filter FWHM. Our results indicate that optimal imaging conditions and reconstruction parameters are necessary to obtain high-resolution images and quantitative accuracy, especially for the high-precision identification of tiny lesions.