Ecosystem provides supporting services, regulating services, provisioning services and cultural services, which have great significance to human well-being and the sustainable development of society. However, the supply and demand of ecosystem services often mismatched in the same region due to the characteristics of spatial and temporal differentiation. Therefore, it is very important for the rational allocation of natural resources when we identify, measure and do spatial-analysis of supply and demand of ecosystem services. Taking Jiaxing City as study area, we used 2005 Landsat7 ETM+ image and 2015 Landsat8 OLI sensor images as the data sources. The ecosystem service matrix method was used to analyze the spatial-temporal differentiation characteristics of supply and demand of the hydrological regulation service (HRS). The following were the analysis results:(1) Jiaxing City was in the stage of rapid urbanization with the significant change of land use/land cover (LULC) from 2005 to 2015, of which constructed areas has increased significantly; water area and paddy area increased, whereas other land area decreased. (2) LULC change was one of the main factors affecting the relationship of supply and demand of the HRS. In this study, we determined that HRS was deficient in most areas of Jiaxing City. (3) With the continuous expansion of built-up areas, the contradiction between supply and demand of HRS has intensified. The shortage supply areas of HRS have expanded from the central city to the urban fringe. (4) Changes in the demand area of HRS were closely related to human activities. The ability of supply areas was subject to the quantity and quality of the natural environment. The flow of ES from supply unit to demand unit was promoted because of the mismatch between the supply and demand of ecosystem services. Policy guidance was important to protect the ecological source, restore and reconstruct the damaged ES flow.