Higher-order harmonics of crowd loads may can lead to an increase in the dynamic response of high-frequency floors, resulting in serviceability and safety issues. This study aims to analyze the effect of different indoor layouts on the human-induced vibration of high-frequency floors. First, a random load model for high-frequency floors is established by combining the social force model (SFM) and a pedestrian load model. Next, a computational model for human-induced vibration of high-frequency floors is developed, taking into account human-structure interaction (HSI). A high-frequency floor with a fundamental frequency of 10.35 Hz is tested to validate the reasonableness of the computational model when applied to different layout configurations. Finally, the serviceability of the floor with different layout forms under random crowd walking conditions is evaluated using the global assessment method for human-induced vibration, with probabilistic results provided. The results show that for human-induced vibration problem in high-frequency floors, the influence of high-order vibration modes must be considered. Under the random walking conditions for five people, the dynamic response of the floor with different layouts is reduced after considering HIS, with a maximum reduction of 13.33% in peak acceleration and a maximum reduction of 12% in probability value of serviceability. The serviceability of the floor varies with the different layout configuration. Specifically, the probability of serviceability problems is highest for the floor with a discussion room layout, followed by the classroom layout, with the meeting room layout the lowest probability.