Disruption of retinal input early in life can lead to amblyopia, a condition characterized by reduced visual acuity after optical correction. While functional abnormalities in the early visual areas have been observed in amblyopia, mesoscale deficits in cortical microcircuitry across cortical depth remain unexplored in humans. Using a combination of submillimeter 7T fMRI and EEG frequency-tagging methods, we investigated neural deficits in monocular processing and binocular interactions in human adults with unilateral amblyopia. The results revealed attenuated and delayed monocular activity in the thalamic input layers of V1, followed by imbalanced binocular suppression and weakened binocular integration in the superficial layers. These disruptions further reduced visual signal strength and processing speed. Our findings pinpoint specific neural deficits in the cortical microcircuitry associated with human amblyopia, offering valuable insights into the mesoscale mechanisms of developmental plasticity and paving the way for more effective treatments for this visual disorder.