In order to study the thickness evaluation of zero-strength layer in the calculation of fire resistance for Cross-Laminated Timber (CLT) floor，the ultimate load-carrying capacity test and fire test of the CLT floors with three and five layers were carried out，respectively. Based on the domestic and foreign wood structure design codes，combined with the simplified design method that ignores the elastic modulus of laminated laminates and only considers the thickness contribution to the moment of inertia of composite sections as well as the reduced cross-section method，a suitable method for the CLT floor was obtained. The variation law of zero-strength layer thickness of CLT floor with charring depth was analyzed. The flexural capacity of CLT floor under high temperature based on the thickness of zero-strength layer in this paper and the results based on EN1995-1-2 were compared. The results showed that the values by the calculation method agreed well with the experimental values. When the charred layer of the CLT floor reached the glue line between cross and longitudinal-layer，the thickness of the zero-strength layer changed continuously；and when the charring depth reached the glue line between longitudinal and cross-layers，the thickness of the zero-strength layer changed sharply. The bending capacity of CLT floor under high temperature obtained by the calculation method in this paper was more reasonable than the prediction of EN1995-1-2，which had better reference value for the fire resistance design of CLT floor in practice.