Nowadays, the comfort of drivers and passengers is drawing more and more attention. The interaction between vehicles and the asphalt road can cause coupled vibration and reduce the comfort of passengers. Therefore, the research on human–vehicle–road coupled vibration and the comfort of passengers is of great importance. In this paper, the three-dimensional human–bus–road coupled vibration system is established, including the bus model, the parallel biomechanical human model with 2 degrees-of-freedom (DOF), and road surface roughness condition. The proposed coupled model was then used to study the dynamic response of the system and the comfort evaluation of the human body. In the comfort evaluation, the annoyance rate based method was proposed to consider the randomness of passenger vibration, the difference of the psychosensory vibration, and the fuzziness of evaluation indicators. Compared to the fuzzy evaluation based on the ISO 2631 standard, the proposed annoyance rate based method gives a quantitative evaluation of human comfort. Not only the degree of comfort can be evaluated, but the percentage of people feeling uncomfortable can also be obtained. Finally, parametric studies were conducted to investigate the effects of road surface roughness, interlayer bonding condition, bus weight, bus speed, and sitting position. It is found that the road surface roughness has the most significant effect on human comfort.