In order to explore the influence of external conditions on the glass transition temperature of asphalt mixture, such as environment and traffic, three factors including heating rate, load frequency, and strain level were considered in this research. Temperature sweep test was developed by DMA method at different test levels. The glass transition temperature of asphalt mixture was obtained by Boltzmann model. The main affecting factors of glass transition temperature were determined by statistical analysis of the experimental results, and then the prediction model of the glass transition temperature under multiple factors was established. Results show that the Boltzmann model could accurately and effectively determine the glass transition temperature of asphalt mixture. The heating rate and load frequency had a significant effect on the glass transition temperature, which increased as the hating rate and load frequency increased. In addition, there was a good linear relationship between the glass transition temperature and the logarithm of heating rate and the load frequency. The glass transition temperature of asphalt mixture had no obvious strain dependence in liner viscoelastic region under bending mechanics condition.