Objective In resting-state functional magnetic resonance imaging (fMRI) study, spontaneous activity patterns abnormally in anisometropic amblyopia can be reflected by the change of amplitude of low frequency fluctuation (ALFF) and the correlation between the amplitude of low frequency fluctuation (ALFF) value and acuity deficit. Methods Twenty two adult anisometropic amblyopes and 21 normal controls participated in this fMRI study. The fMRI data were analyzed to get ALFF value in each subject. Two sample t test was carried out to analysis ALFF value within the whole brain for the inter groups. The correlation between the ALFF value and the amblyopic eye’s visual acuity was calculated as the Pearson correlation coefficient. Results The ALFF value mainly increased inferior temporal gyrus, superior temporal gyrus and occipital lobe, while decreased in cerebellum posterior lobe, inferior frontal gyrus and superior frontal gyrus. Further the changes in ALFF such as cerebellum posterior lobe, precuneus, occipital lobe, superior temporal gyrus and frontal lobe had significant correlation with amblyopia acuity deficit. Conclusions ALFF is an effective noninvasive algorithm, which can help to exploring spontaneous patterns changes in amblyopia. And multiple area existed patterns abnormal and some of them may relate to the acuity deficit. These results have important implications for models of amblyopia and approaches to treatment.