Eye-tracking technology has been widely applied to various kinds of traditional sports research, such as football, basketball and table tennis. eSports is an emerging digital sport, which is currently enjoying rapid development. The previous study primarily focuses on the practical applications of eye movement in eSports, such as game design, visual attention, and performance analysis. However, there is a scientific gap for the specific differences between expert and novice players, the potential of eye-tracking as a biological marker for eSports performance, and the transfer of insights from real sports to eSports through eye-tracking studies. This study aims to uncover disparities in eye movement characteristics between professional players (N = 14, 24.1 ± 2.5 years) and non-professional players (N = 14, 25.2 ± 2.3 years) by employing the expert-novice paradigm. We extracted several key eye movement features, including average fixation counts, average fixation duration, first fixation duration, eye movement trajectory, and visits to areas of interest. The results indicate that professional players exhibit higher fixation counts, shorter first fixation duration, reduced average fixation duration, and employ a more effective visual search strategy. Professional players’ fixation trajectories are characterized by clarity and focus, with a greater number of fixation points concentrated on key areas of interest. In contrast, non-professional players demonstrate relatively random fixation trajectories, lacking distinct focal points. This study not only advances our understanding of eye-tracking in eSports but also provides technical guidance and practical significance for further research in this burgeoning field.