Water resources in the soil play an essential role in hydrological processes and ecosystem functions on the Tibetan Plateau. However, accurately measuring soil moisture distribution in this region presents challenges due to the diverse ecosystem types, complex terrain, and harsh environmental conditions. In this study, we introduce an approach for estimating mesoscale soil moisture in the Qilian Mountains (QLM) region of the eastern Tibetan Plateau using a cosmic-ray neutron rover. Soil moisture estimates derived from neutron count rates, newly adjusted by vegetation effects, demonstrated good agreement with soil moisture measurements obtained through soil sampling at 26 calibration sites across the region (RMSE = 0.025 g g−1). The calibration parameter N0_NDVI was 443 cpm in the QLM. Utilizing NDVI as vegetation correction method showed potential improvements in the accuracy of converting neutron counts to soil moisture across the diverse mountainous ecosystem types. The newly developed calibration equation provided a high-precision, high spatial resolution soil moisture transect across various landscapes measured by the rover. The average mesoscale soil moisture along the rover route varied by ecosystem types, with values of 0.10 g/g in deserts, 0.17 g/g in grasslands, 0.13 g/g in forests, 0.18 g/g in subalpine shrublands, and 0.20 g/g in croplands. Land cover types emerged as crucial determinants of mesoscale soil moisture variability in the QLM region. These findings offer valuable mesoscale soil moisture data and new insights into soil water information at the transect scale across diverse ecosystem types in the Tibetan Plateau.