A stress group should be subdivided into eustress (low-stress) and distress (high-stress) groups to better evaluate personal cognitive abilities and mental/physical health. However, it is challenging because of the inconsistent pattern in brain activation. We aimed to ascertain the necessity of subdividing the stress groups. The stress group was screened by salivary alpha-amylase (sAA) and then, the brain’s hemodynamic reactions were measured by functional near-infrared spectroscopy (fNIRS) based on the near-infrared biosensor. We compared the two stress subgroups categorized by sAA using a newly designed emotional stimulus-response paradigm with an international affective picture system (IAPS) to enhance hemodynamic signals induced by the target effect. We calculated the laterality index for stress (LIS) from the measured signals to identify the dominantly activated cortex in both the subgroups. Both the stress groups exhibited brain activity in the right frontal cortex. Specifically, the eustress group exhibited the largest brain activity, whereas the distress group exhibited recessive brain activity, regardless of positive or negative stimuli. LIS values were larger in the order of the eustress, control, and distress groups; this indicates that the stress group can be divided into eustress and distress groups. We built a foundation for subdividing stress groups into eustress and distress groups using fNIRS.