PURPOSE Impairment of visual acuity (VA) can be seen early on in various diseases and has a major impact on patients' daily activities. Zebrafish is an important model for studying visual disorders. We developed a new method in zebrafish larva to easily and precisely measure the VA, which should allow for better estimation of affected vision such as after genetic manipulation or pharmacologic intervention. METHODS We used an optokinetic reflex (OKR) paradigm with a staircase technique to estimate VA of zebrafish larva. Consistent eye displacements were used as the indicator for OKR. We measured VA and determined the dependence of VA on clockwise and counterclockwise horizontal stimulus directions. RESULTS Visual acuity in zebrafish larva was estimated to be 0.179 ± 0.013 cyc/deg binocularly and 0.129 ± 0.008 cyc/deg (left eye) and 0.128 ± 0.012 cyc/deg (right eye) monocularly. We found within single subjects spatial frequency thresholds that showed highly significant differences between the two horizontal stimulus directions. Average higher and lower binocular thresholds were 0.181 ± 0.026 and 0.158 ± 0.014 cyc/deg, respectively. Importantly, no correlations were found between spatial frequency thresholds and average median peak slow-phase eye velocities (SPV) of OKR in all experiments. CONCLUSIONS Consistent eye displacements evoked by OKR stimuli can be used as an indirect measure of VA in zebrafish larva. Conversely, using SPV of OKR to determine VA does not seem to be accurate. With our method, single larva showed significantly different VA depending on stimulus directions, which might reflect asymmetric maturation of retinal and/or visual pathway structures.