Objective To investigate the characteristics of eye movement in children with anisometropic amblyopia, and to compare those characteristics with eye movement in a control group. Methods 31 children in the anisometropic amblyopia group (31 amblyopic eyes in group A, 31 contralateral eyes in group B) and 24 children in the control group (48 eyes in group C). Group A was subdivided into groups Aa (severe amblyopia) and Ab (mild-moderate amblyopia). The overall age range was 6-12 years (mean, 7.83 +/- 1.79 years). All children underwent ophthalmic examinations; eye movement parameters including saccade latency and amplitude were evaluated using an Eyelink1000 eye tracker. Data Viewer and MATLAB software were used for data analysis. Results Mean and maximum saccade latencies, as well as mean and maximum saccade amplitudes, were significantly greater in group A than in groups B and C before and after treatment (P < 0.05). Mean and maximum saccade latencies were significantly different among groups Aa, Ab, and C (P < 0.05). Pupil trajectories in two detection modes suggested that binocular fixation was better than monocular fixation. Conclusions Eye movement parameters significantly differed between contralateral normal eyes and control eyes. Clinical evaluation of children with anisometropic amblyopia should not focus only on static visual acuity, but also on the assessment of eye movement.