<p>This study investigated the crosslinking behavior and curing properties of environmentally friendly polyurethane (PU) modified asphalt (PUMA) prepared by in-situ polymerization. The research aimed to clarify the chemical reaction sites and sequence between the PU prepolymer (PUP) and asphalt components, and to evaluate the microstructural and performance evolution of PUMA during curing. Density functional theory (DFT) calculations were employed to predict reactive sites based on electrostatic potential and frontier molecular orbitals, and to compute Gibbs free energy and transition state energy barriers. The experimental results indicate that phenolic hydroxyl and pyridinic nitrogen in asphaltene molecules and phenolic hydroxyl in resin molecules are the primary sites for reaction with PUP. PUP reacts spontaneously with chain extender (BDO), water, asphaltene, and resin at room temperature, with the reaction following the order: BDO &gt; H₂O &gt; asphaltene-1 &gt; asphaltene-2 &gt; resin. Microstructural analysis by atomic force microscopy reveals that PU acts as a bridge connecting asphaltene and resin, forming an integrated network that became more perfect with prolonged curing time. Fourier transform infrared spectroscopy of SARA fractions confirms the formation of amide bonds between PUP and asphalt components. The high-temperature performance of PUMA improves with curing time, while the low-temperature performance slightly decreases but still meets specification requirements after 180 days of curing. The findings can provide a fundamental basis for the design and preparation of high-performance PUMA.</p>