The spatial relationships between traffic accessibility and supply and demand (S&D) of ecosystem services (ESs) are essential for the formulation of ecological compensation policies and ESs regulation. In this study, an ESs matrix and coupling analysis method were used to assess ESs S&D based on land-use data for 2000, 2010, and 2020, and spatial regression models were used to analyze the correlated impacts of traffic accessibility. The results showed that the ESs supply and balance index in the middle reaches of the Yangtze River urban agglomeration (MRYRUA) continuously decreased, while the demand index increased from 2000 to 2020. The Gini coefficients of these indices continued to increase but did not exceed the warning value (0.4). The coupling degree of ESs S&D continued to increase, and its spatial distribution patterns were similar to that of the ESs demand index, with significantly higher values in the plains than in the montane areas, contrasting with those of the ESs supply index. The results of global bivariate Moran's I analysis showed a significant spatial dependence between traffic accessibility and the degree of coupling between ESs S&D; the spatial regression results showed that an increase in traffic accessibility promoted the coupling degree. The present results provide a new perspective on the relationship between traffic accessibility and the coupling degree of ESs S&D, representing a case study for similar future research in other regions, and a reference for policy creation based on the matching between ESs S&D in the MRYRUA.