Abstract: The rapid advancement of microfluidic technology has provided essential support for biomedical and related applications, in which the migration and conformational evolution of polymer chains within microchannels are critical for improving the stability and accuracy of sample detection. To address the complex dynamic behavior of polymer chains in microscale confined spaces, the present study employs the mesoscale dissipative particle dynamics (DPD) method to investigate single-chain transport in contraction–expansion microchannels. An improved finite extensible nonlinear elastic (FENE) polymer chain model was constructed, and the effects of chain stiffness coefficient, contraction–expansion ratio, and Reynolds number on the radius of gyration and the center-of-mass migration behavior of a single polymer chain were systematically examined. The results reveal that the polymer chain exhibits a distinct two-stage conformational evolution as it traverses the contraction region, characterized by pronounced inlet stretching followed by outlet recoiling. On this basis, a quantitative correlation between the enhancement of chain stiffness and conformational extension was established. The polymer chain predominantly adopts a coiled conformation in the expansion region of the microchannel, whereas an elongated conformation prevails in the contraction region. A decrease in the contraction–expansion ratio further intensifies the spatial confinement effect and increases the maximum elongation of the polymer chain. By contrast, an increase in the contraction–expansion ratio alleviates the resistance to conformational rearrangement, thereby improving the transport efficiency of the polymer chain through the channel. Meanwhile, the center of mass of the polymer chain exhibits an overall center-enriched distribution; however, as the Reynolds number and stiffness coefficient increase, it progressively deviates from the channel centerline and displays a pronounced tendency toward near-wall migration. These findings deepen the understanding of polymer chain transport and conformational evolution under microfluidic confinement, and provide a theoretical foundation for the efficient detection of biomacromolecules and the structural optimization of microfluidic devices.