Dissolved organic matter (DOM) is ubiquitous in aquatic environment and is of great significance in various biogeochemical cycling processes. Rapid urbanization coupled with intensified human activities has significantly altered the content and characteristics of DOM in surface water at watershed. It has importance of characterize the spatial distribution of DOM at the molecular level for effective watershed management. In this study, the spatial variation of molecular characteristics of DOM as well as its influent factors was analyzed in a typical peri-urban watershed (Zhangxi watershed) in the Yangtze River Delta. Water samples were collected in July 2022 from sites from various land use and intensity of human activities. Results showed that the molecular characteristics of DOM were dominated by CHO type compounds, with lignin/carboxylic-rich alicyclic molecules (CRAM)-like structures as the main proportion. Significant higher content of CHON and CHONS compounds were observed in sub-watersheds with higher intensive human disturbance. The intensity of human activities was indirectly assessed with three indices: distance to town (DT), distance to water source (DS) and proportion of land use types. Our results showed that human activities played a significant role in the spatial distribution of heteroatoms of DOM. A positive correlation was observed between DS and both the intensity weighted average of nitrogen atoms (N_wa) and the percentage of CHON compounds of DOM molecules, while a negative correlation was found with the intensity weighted average of carbon and hydrogen atoms, as well as the ratio of charge to mass (C_wa, H_wa, m/z_wa). The relative abundance of CHON-containing formulae was positively correlated to the proportion of agricultural land use whereas negatively correlated to the proportion of forest. Compared with the DOM in typical land use types: unique CHON molecules exhibited relatively higher abundances in farmland and urban land use types than that in forest land. The CHON molecular formula was enriched in the lignin/CRAM-like structures region of the water samples in farmland land use types. The CHON molecular formula was enriched in fat and lignin/CRAM-like structures region of the water samples in urban land use types. In short, our results revealed that anthropogenic activities significantly altered the molecular characteristics of surface water DOM in peri-urban watersheds, augmenting molecular diversity and facilitating the enrichment of specific molecular formulas.