Human activity factors have a significant impact on changes in ammonia nitrogen (NH3-N) content in rivers. Existing research mainly focuses on human activity factors as type factors, and lacks research on the key factors affecting river NH3-N among human activity factors. Therefore, this paper aims to study the key factors affecting human activities on NH3-N in the Huaihe River through various statistical analysis methods. The study found that changes in NH3-N content in the Huaihe River are mainly affected by land use patterns in the basin. There are two different ways in which land use affects NH3-N in rivers: direct effects and indirect effects. We also studied the main pathways through which changes in key factors in human activities affect NH3-N in the Huaihe River by constructing a structural equation model. The results showed that crop sowing area and afforestation area have a significant direct effect on NH3-N in the Huaihe River. In addition, crop sowing area and afforestation area can also affect river NH3-N by regulating the amount of nitrogen fertilizer and human excrement. This study is of great significance for understanding how human activities regulate NH3-N content in rivers.