Accurate estimation of route flow is crucial to traffic control optimization. Existing route flow estimation models are typically built upon strong assumptions on traveler route choice behavior or turn to data-driven approaches which heavily rely on prior OD estimation and high sensor coverage rate. This study proposes a route flow estimation framework by fusing the probe vehicle trajectory and automated vehicle identification (AVI) data. The notion of probe vehicle route penetration rate (RPR) is utilized for the construction of a novel data-driven objective function. Sensor-OD flow and observable route flow penetration are formulated as model constraints. The solution uniqueness is guaranteed though an iterative procedure under the principle of entropy maximization. Numerical studies are conducted based on pNEUMA, a large-scale full sample trajectory dataset. Results show that the proposed model performs better than the traditional path flow estimation model, especially when the prior flow information is not available. Sensitivity analysis on AVI sensor coverage rate, probe vehicle route coverage rate and RPR distribution demonstrates the proposed model’s applicability in practice.