The design of timber-concrete composite (TCC) floors is usually subject to the requirement of serviceability rather than strength. Most studies reported so far focus on the dynamic characteristics of the TCC floor. However, there are few studies conducted on the vibration comfort of TCC floors under human-induced excitation. In this paper, dynamic experiments were performed to obtain the fundamental frequency and the damping ratio of a glulam-concrete composite floor under simply supported and fixed support boundary conditions, respectively. Besides, an in-depth investigation was carried out considering the influences of walking path, step frequency, number of pedestrians, arrangement of walking loads and walking mode on the vibration response of the composite floor. Test results showed that the peak acceleration of the floor increased with an increase in step frequency and the number of pedestrians. Regular walking caused a higher peak acceleration compared to irregular walking. At the same step frequency, marking time caused a larger peak acceleration than normal walking. Three existing analytical models were applied to predict the fundamental frequency of the floor, which conformed to the experimental results. In addition, a calculation model and finite element (FE) models were proposed to predict the peak floor acceleration under a single person walking, and the numerical model results agree well with the test results. A double-index was proposed to evaluate the vibration serviceability of TCC floors; the fundamental frequency of the floor is required to exceed 15 Hz and the peak acceleration ought to be less than 0.15 m/s2.