Focusing on the fire risk of the horizontal multi-layer cable tray in a nuclear power plant, the characteristics of the smoke layer temperature profile generated by the fire on the multi-layer cable tray in a closed compartment and the prediction model for the smoke layer temperature were investigated. Fire experiments of a three-layer cable tray with two typical cable arrangements were conducted in a closed compartment. The experimental results showed that the stratification of vertical smoke layer temperature was evident. Based on the vertical temperature profile in the compartment center, the thermal environment resulted from the fire can be classified as top ceiling jet layer, middle smoke layer, and lower cool air layer. The unsteady-state temperature prediction model in a closed compartment was used to predict the smoke layer temperature resulted from the fire on the horizontal multi-layer cable tray. The comparison of the predicted results with the experimental temperature for the smoke layer showed that this model can accurately predict the maximum temperature of the smoke layer, with relative error less than 1%. the prediction model underestimated the temperature generated by the decay stage of the cable tray fire, and thus the global error is within 16.3% and 27.8%.