In this paper, the nonlinear stiffness of a polyester rope under dynamic loads at different temperatures was experimentally investigated. The effects and evolution of the average load, load period and load amplitude on dynamic stiffness were studied. The experimental results show that the dynamic stiffness at 30 °C was greater than that at 20 °C and 40 °C. This is because the dynamic stiffness reduced as the temperature and humidity increased, while in the closed thermostat, humidity decreased with an increase in temperature. In response to this phenomenon, a new set of empirical equations was proposed to consider temperature, humidity, and dynamic load based on the existing empirical formulas, by combining the experimental results. A set of experiments was conducted at 25 °C to verify the accuracy and applicability of the formula. The experimental results and the predicted results are in good agreement.