Understanding the travel flux between urban blocks is fundamental for traffic demand prediction, urban area planning and urban traffic management. However, the uncertainty of human mobility patterns and the complexity of urban transportation systems usually yield challenges in accurately estimating the travel flux within a city. Thus, we propose a novel travel flux estimation method that integrates traffic flow characteristics (traffic volume and travel time), spatio-temporal autocorrelation, and travel purpose correlation. First, the geographically weighted method was used to model and verify the spatio-temporal autocorrelation of origin–destination flows, whereas the purpose correlation of origin–destination flows was expressed through the function feature vectors of urban blocks. Then, a multi-objective bi-level programming model, according to the generalized least squares method, was constructed to estimate the travel flux between blocks. This was used to solve the problem of accurate estimation of travel flux by combining microscopic traffic flow characteristics with macroscopic spatio-temporal and purpose characteristics. Finally, an empirical analysis of the Hankou district, Wuhan City, demonstrated that in contrast to the existing method, the accuracy of the proposed method for predicting the human travel flux improved by approximately 20%. The estimated results were consistent with the spatial distribution pattern of human travel. Moreover, these results can provide targeted decision support for planning urban spaces, allocating urban resources, and guiding vehicular traffic.