Perceiving emotions from the movements of other biological entities is critical for human survival and interpersonal interactions. Here, we report that emotional information conveyed by point-light biological motion (BM) triggered automatic physiological responses as reflected in pupil size. Specifically, happy BM evoked larger pupil size than neutral and sad BM, while sad BM induced a smaller pupil response than neutral BM. Moreover, this happy over sad pupil dilation effect is negatively correlated with individual autistic traits. Notably, emotional BM with only local motion features retained could also exert modulations on pupils. Compared with intact BM, both happy and sad local BM evoked stronger pupil responses than neutral local BM starting from an earlier time point, with no difference between the happy and sad conditions. These results revealed a fine-grained pupil-related emotional modulation induced by intact BM and a coarse but rapid modulation by local BM, demonstrating multi-level processing of emotions in life motion signals. Taken together, our findings shed new light on BM emotion processing, and highlight the potential of utilizing the emotion-modulated pupil response to facilitate the diagnosis of social cognitive disorders.