While amblyopia involves both binocular imbalance and deficits in processing high spatial frequency information, little is known about the spatial-frequency dependence of binocular imbalance. Here we examined binocular imbalance as a function of spatial frequency in amblyopia using a novel computer-based method. Binocular imbalance at four spatial frequencies was measured with a novel dichoptic letter chart in individuals with amblyopia, or normal vision. Our dichoptic letter chart was composed of band-pass filtered letters arranged in a layout similar to the ETDRS acuity chart. A different chart was presented to each eye of the observer via stereo-shutter glasses. The relative contrast of the corresponding letter in each eye was adjusted by a computer staircase to determine a binocular Balance Point at which the observer reports the letter presented to either eye with equal probability. Amblyopes showed pronounced binocular imbalance across all spatial frequencies, with greater imbalance at high compared to low spatial frequencies (an average increase of 19%, p < 0.01). Good test-retest reliability of the method was demonstrated by the Bland-Altman plot. Our findings suggest that spatial-frequency dependent binocular imbalance may be useful for diagnosing amblyopia and as an outcome measure for recovery of binocular vision following therapy.