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Heart rate variability (HRV) analysis is commonly used as a quantitative marker depicting the activity of autonomic nervous system (ANS) that may be related to mental or psychological stress. HRV features can be extracted by detecting QRS complexes from electrocardiogram (ECG) signals. The purpose of this paper is to assess the biological effect of visual stimulus in terms of autonomic nervous activity. A non-invasive multi-channel ECG monitoring system has been designed and implemented to investigate the differences in the heart rate variability (HRV) of individuals during different visual stimulus. HRV analysis revealed increased parasympathetic nervous activity while perceiving the positive visual stimulus, as well as a relative reduction in HRV and cardiac vagal tone in negative visual stimulus, and long term negative visual stimulus may weak the function of ANS which affect our body detrimentally.