Agricultural non-point source pollution (AGNPS) threatens sustainable agricultural development; the impact of land fragmentation (LF) on AGNPS has been a concern for local managers. Based on agricultural production, livelihoods, and land use data from 2010 to 2020 in the Three Gorges Reservoir Area (TGR), this paper first measured the sources of AGNPS emissions. Second, it constructed the evaluation index system of land fragmentation. Then, the paper explored the relationship and impact intensity of LF on AGNPS. The results showed that: (1) the TGR central region was the high-value area of AGNPS production, while pollution emissions showed a decreasing trend between 2010 and 2020. COD showed the largest decrease of 21.85%. (2) Land use index (LUI) and aggregation index (AI) are two indicators for evaluating LF. In Chongqing, the LUI value is higher in the main urban area, while the AI value around the main urban area is higher. (3) LUI was negatively correlated with pollutants, while AI was positively correlated. Among the contributions to COD, the share of LUI was 21%, and AI was 13.24%. The results can be used to analyze the situation of AGNPS and land fragmentation and provide targeted policy suggestions for AGNPS control.