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Due to the unique hydrogeological environment of karst areas, pollutants are more likely to enter the soil and water, showing a special migration and transformation behavior. In this work, the binding behaviors between strontium (Sr²⁺) and dissolved organic matter (DOM) extracted from soil under the influence of pH and Ca²⁺ in a typical karst area were investigated by applying three-dimensional fluorescence spectroscopy combined with parallel factor analysis (EEM–PARAFAC) and two-dimensional correlation analysis (2D-COS) of synchronous fluorescence spectra (SF). The results show that DOM extracted from soil was dominated by tryptophan-like and tyrosine-like materials (77% in total). Two-dimensional COS of SF showed that the tryptophan-like substance in DOM extracted from soil preferentially bound to Sr²⁺. When the pH was 7, the binding coefficient (log<i>K_a</i>) of the four DOM components ranged from 2.69 to 4.04, which was more conducive to the binding of DOM extracted from soil and Sr²⁺ than under acidic and alkaline conditions. Ca²⁺ in soil weakened the binding of DOM extracted from soil to Sr²⁺ by competing for binding sites and changing the molecular surface potential. This research is helpful for acknowledging the migration and transformation of Sr²⁺ and offers a reference for groundwater protection in karst areas.