The environment is one of the factors that may influence occupants’ perception of floor vibration and the assessment of floor serviceability. In this study, laboratory tests were conducted on a 3-ply CLT floor. Occupants’ assessment of the floor serviceability under human-induced vibration was investigated. Virtual reality (VR) technique was used as a research method, simulating two common environments in life. First, the correlation between the occupants’ annoyance rating and serviceability indicators (response factor and vibration dose value (VDV)) was compared with existing standards. The results show that the response factor method in ISO 10137:2007 is conservative for timber floors in both bedroom and gym environments. The VDV method in BS 6472-1:2008 can generally reflect the vibration acceptability of timber floor vibration. Then, the effect of acceleration and environment on the floor serviceability assessment was investigated through statistical methods, respectively. A weak positive correlation between the annoyance rating and the acceleration was found. The effect of the environment on floor vibration assessment was found to be significant.