More than 60% of the world’s ecosystem services have deteriorated over the past few decades. Studying the spatio-temporal fluctuations in ecosystem service value and its influencing factors is important for identifying regional ecosystem service value issues, upholding regional ecological harmony, and encouraging regionally healthy and coordinated sustainable development. Ecosystem service value has so far been studied primarily in relation to the effect of socioeconomic and physical–geographical variables. However, the trade-offs and synergies among ecosystem service values also drive the spatio-temporal variations in ecosystem service value. Few studies have been conducted to date to investigate the trade-offs and synergies between ecosystem service values and their impact on ecosystem service value. Therefore, this paper used sensitivity analysis, correlation analysis, trade-offs and synergies analysis, and a Geodetector to examine changes in ecosystem service value and their influencing factors within the Nanjing metropolitan region. The ecosystem service value decreased somewhat overall between 2000 and 2020, with a decline rate of 2.19 million CNY/year. In comparison to the north of the Nanjing metropolitan region, the ecosystem service value was relatively higher in the south. The water bodies had the highest total ecosystem service value, followed by forest land, cultivated land, and grassland, with construction land and unused land having the lowest ecosystem service values overall. The main socioeconomic factor influencing the spatial variations in ecosystem service value was population density, while the main physical–geographical factors were the digital elevation model, the normalized difference vegetation index, and precipitation. As a result, the Nanjing metropolitan area should tighten its grip on excessive population growth. In contrast to the expository strength of a single factor on the ecosystem service value, the influence of all individual elements on the ecosystem service value under interaction was significantly increased, and the interaction among the normalized difference vegetation index and gross economic product had the most obvious effect on the ecosystem service value. The spatial variation in the ecosystem service value was also influenced by trade-offs and synergies between the value of supply services, regulation services, support services, and cultural services. Therefore, trade-offs and synergies among ecosystem services also need to be considered in land-use decisions.