Many neuroimaging tools have been used to assess the site of the cortical deficits in human amblyopia. In this paper, we aimed at detecting the structural and functional deficits in humans with amblyopia, with the aid of anatomic magnetic resonance imaging (aMRI) and functional MRI (fMRI). We designed the visual stimulus to investigate the functional deficits, and delineated the V1/V2 areas by retinotopic mapping. Then we performed the brain parcellation to calculate the volume of the subcortical structure on each individual, and reconstructed the cortical surfaces to measure the cortical thickness. At last, the statistical comparison was carried out to find the structural abnormities and their relationship to the functional deficits. Compared with the normal controls, it is found that the hemisphere difference existed on the unilateral amblyopia subjects, and the functional deficit might come along with the changes in the cortical volume, especially in the occipital lobe. The examined results may provide insight to the study of the neural substrates of amblyopia.