To detect the altered spontaneous brain activity patterns in children and adults with anisometropic amblyopia using resting-state functional magnetic resonance imaging (rs-fMRI) technique combined with the amplitude of low-frequency fluctuation (ALFF) method. Thirty-two monocular anisometropic amblyopia and 34 normal-sight controls were divided into child group and adult group. Rs-fMRI was performed in all participants and analysis of ALFF value within the whole brain was conducted in each subject. ALFF value differences between the patients and controls in the two groups were compared via an independent two-sample t test. The amblyopic children mainly exhibited increased ALFF in part of the bilateral calcarine (BA17), the left middle occipital gyrus (BA18/19), and the left postcentral gyrus (BA2). By contrast, the amblyopic adults showed decreased ALFF in the bilateral precuneus cortex (part of BA7), and the standardized ALFF value of bilateral precuneus were correlated with the amount of anisometropia of the amblyopic adults. Rs-fMRI is an effective noninvasive technique for exploring brain activity of the anisometropic amblyopia. Our findings demonstrated that brain activity changed both in amblyopic children and adults under the resting state, and revealed the differences in spontaneous activity patterns between the amblyopic children and adults.