Fluorescence excitation-emission matrix spectroscopy (EEMs) has become a very popular technique in characterization of aquatic dissolved organic matter (DOM) coupled with a parallel factor (PARAFAC) model, denoted as (EEMs-PARAFAC). This research addresses the poorly researched relationship correlation between dissolved ions and fluorescence in a natural water environment. The relationship between the EEMs-PARAFAC components and ionic composition was studied in freshwater lakes, rivers, and seawater from locations in China. The natural water environment is different from a simulated environment having a fixed ionic composition. We used electrical conductivity (EC) to reflect the ionic strength as an indicator to evaluate the relationship in a series of water bodies. Results show that the EC generally had a positive correlation with DOM in natural water environment, but no correlation was found with water from the highly saline Yellow Sea. The Chaohu Lake samples contained one component having a significant negative correlation with EC, i.e., r > 0.6, p < 0.05, while other surface waters contained components having both positive and negative correlations (r > 0.5, p < 0.05). The negative correlation with EC also highlighted that humic acid-like components and protein-like materials (c1-c3) were positively correlated with DOM, while the protein-like component (c4) was negatively correlated with DOM. The EC equation proposed provided a good fit with the EC values of surface waters. The use of EC would be a useful and rapid method for analyzing the variation in the fluorescence component and its effect on water quality. This study highlights the need to account for variation in EC when assessing EEMs-PARAFAC of natural waters.