Abstract: Ecosystem services (ESs) are crucial for human survival and development, and changes in ESs are closely linked to both the natural environment and human activity. As counties have become the fundamental unit of China’s new urbanization, investigating the dynamic processes of ESs, the driving mechanisms, and their coupling with urbanization at the county scale holds significant theoretical and practical value. However, further research is still needed to analyze the impact persists regarding the analysis of county-scale urbanization effects on the ecological environment and the coupling relationship between the two. This study measured the spatio-temporal characteristics of ESs and urbanization level (UL) in the Yellow River Basin (YRB) from 2000–2020 at county scale, utilizing multi-period land use, meteorological, and statistical data. Subsequently, the research investigated the effects of anthropogenic and natural factors on comprehensive ecosystem services (CES), as well as the coupling relationship between the UL and CESs. The results revealed fluctuating ES levels across different periods, characterized by a general upward trend. Geographically, lower levels were observed in the eastern plain and western desert areas, while higher levels were observed in the central transition area. From 2000–2020, the impervious surface area, population, and gross domestic product (GDP) of the YRB increased consistently. The UL decreased gradually from the southeast to the northwest, with high levels concentrated in city centers. Among anthropogenic factors, population agglomeration and land development significantly influenced CES, with their impact intensifying over time. Slope and elevation were identified as the primary natural factors affecting CES. During the study period, the overall coupling coordination degree of the UL and CES in each county increased, albeit with notable spatial disparities. Regarding synchronization types, the number of downstream counties experiencing ecological losses increased. This study provides insights for managing the relationship between urbanization and ecosystems and offers a framework to migrate the conflict between regional socioeconomic development and the natural environment in the YRB.