The transmission risk of airborne diseases in public transportation systems is a concern. This paper proposes a modified Wells-Riley model for risk analysis in public transportation systems to capture the passenger flow characteristics, including spatial and temporal patterns, in the number of boarding and alighting passengers, and in number of infectors. The model is used to assess overall risk as a function of origin–destination flows, actual operations, and factors such as mask-wearing and ventilation. The model is integrated with a microscopic simulation model of subway operations (SimMETRO). Using actual data from a subway system, a case study explores the impact of different factors on transmission risk, including mask-wearing, ventilation rates, infectiousness levels of disease, and carrier rates. In general, mask-wearing and ventilation are effective under various demand levels, infectiousness levels, and carrier rates. Mask-wearing is more effective in mitigating risks. Impacts from operations and service frequency are also evaluated, emphasizing the importance of maintaining reliable, frequent operations in lowering transmission risks. Risk spatial patterns are also explored, highlighting locations of higher risk.