Amblyopia is a visual development deficit caused by abnormal visual experience in early life, mainly manifesting as defected visual acuity and binocular visual impairment, which is considered to reflect abnormal development of the brain rather than organic lesions of the eye. Previous studies have reported abnormal spontaneous brain activity in patients with amblyopia. However, the location of abnormal spontaneous activity in patients with amblyopia and the association between abnormal brain function activity and clinical deficits remain unclear. The purpose of this study is to analyze spontaneous brain functional activity abnormalities in patients with amblyopia and their associations with clinical defects using resting-state functional magnetic resonance imaging (fMRI) data. In this study, 31 patients with amblyopia and 31 healthy controls were enrolled for resting-state fMRI scanning. The results showed that spontaneous activity in the right angular gyrus, left posterior cerebellum, and left cingulate gyrus were significantly lower in patients with amblyopia than in controls, and spontaneous activity in the right middle temporal gyrus was significantly higher in patients with amblyopia. In addition, the spontaneous activity of the left cerebellum in patients with amblyopia was negatively associated with the best-corrected visual acuity of the amblyopic eye, and the spontaneous activity of the right middle temporal gyrus was positively associated with the stereoacuity. This study found that adult patients with amblyopia showed abnormal spontaneous activity in the angular gyrus, cerebellum, middle temporal gyrus, and cingulate gyrus. Furthermore, the functional abnormalities in the cerebellum and middle temporal gyrus may be associated with visual acuity defects and stereopsis deficiency in patients with amblyopia. These findings help explain the neural mechanism of amblyopia, thus promoting the improvement of the treatment strategy for amblyopia.