Understanding the impact of land use/cover change on urban flood regulation ecosystem service (UFRES) is important for improving the urban ecosystem services and urban stormwater management. In this study, Landsat images and hybrid classification approach (including linear spectral unmixing and artificial visual interpretation) were applied to extract urban land use/cover data of Beijing from 1991 to 2014. Then, urban surface runoff and UFRES under different rainfall returning periods were simulated and evaluated in built-up areas, respectively. And we further analyzed the impact of land use/cover change on UFRES. The results indicated that the total area of built-up land increased by 1096.53 km² from 1991 to 2014 in Beijing. Meanwhile, urban impervious surface area (ISA) fraction increased by 9.83%. The UFRES showed a decreased trend with a decrease of runoff reduction efficiency by 5.37%, 2.40%, 1.75% and 1.04% under 1a, 10a, 25a and 100a rainfall returning periods, which increased urban flood risk in a certain extent. Comparing with the old urban area, the land cover change had more significant impact on UFRES in the new expansion urban area, and its contribution ratio is higher than 84%. We suggest the fraction of urban ISA and green space(GS) should be designed properly in the future urban planning to improve UFRES.