With the development of social economy and the improvement of people’s living standard, the building with large-span floor has gradually developed. The vertical natural vibration frequency of floor structures is getting closer to the load frequency of human activities, which may generate dynamic response affecting comfort. Therefore, the vibration comfort problem in the building floor has begun to attract people’s attention. The comfort problem needs to be considered in the structural design stage. If the vibration comfort problem of the floor structure is found after the completion of the project, it is difficult and costly to strengthen. In this paper, the structure of large-span floor system in rehearsal room was taken as the engineering background, The frequency of the first order overall vertical mode of the large-span floor structure is low, which is close to the vibration load frequency generated by the users of the rehearsal hall, and the floor is prone to produce dynamic responses that affect comfort. Therefore, the vibration comfort control was conducted in the structural design stage for the large-span floor structure of the rehearsal hall, according to the current national standard JGJ/T 441-2019 Technical Standard for Human Comfort of the Floor Vibration. Firstly, through modal analysis, the dynamic characteristics of the floor system, the location of the maximum vibration point and the time history curves of the excitation loads were determined. Secondly, through dynamic response analysis, the comfort of various vibration load conditions was evaluated, and the controlling vibration load condition for the comfort of the floor system in rehearsal room was determined. When no vibration reduction measures are taken, under the control condition of crowd dancing excitation, the steady-state peak acceleration of the most unfavorable vibration point of the floor is as high as 1.047m/s2, and the dancers will obviously feel uncomfortable. Lastly, the vibration reduction design for the floor system was conducted by means of tuned mass damper (TMD). The influence of TMD’s mass, damping ratio and frequency ratio on the vibration reduction ratio was studied. The peak acceleration of the maximum vibration point of the floor system during the steady-state vibration stage was successfully controlled to 0.4837m/s2(within the limit of standard), and the vibration reduction rate is 53.8%, which meets the requirements of the dancing crowd for comfort.