Despite of an important concern, human bioaerosol emission into subway is not well and directly characterized. Here, we used bioaerosol detector and next generation sequencing methods to investigate time-dependent bioaerosol size distributions in Beijing subway system between March and April, 2015. In contrast to weekends, weekday microbial aerosol results exhibited strong time dependence with higher bacterial and fungal aerosol levels up to 2083 CFU m–3 and 483 CFU m–3 observed, respectively, for the peak hours. During the peak hour (17:30–18:30), bioaerosol emissions at 0.8–3 µm was detected, while about three times higher concentration levels were observed compared to those during the off-peak hour (22:00–23:00). Similar bioaerosol size distributions were observed between ventilation outlets and subway platform air. During off-peak hours, subway bioaerosols had similar size distributions with the outside air. Sequence results revealed a vast array of airborne microbial species which varied from one station to another, but with Aspergillus spp. as dominant fungal species, and Staphylococcus, Pseudomonas as primary bacterial genera including human opportunistic ones. Our results provide direct online observations of human contributions to subway size-resolved bioaerosols, and enhancing ventilation system might help for controlling the exposure especially during the peak-hours.