In this study, disinfection by-products (DBP) formation from dissolved organic matter (DOM) and its fractions, including both hydrophilic and hydrophobic components, were investigated at a typical karst surface water. The subsequent DBP formation potential was evaluated by deducing chemical characteristics of DOM fractions and representative algal organic matter (Chlorella sp. AOM) under the influence of divalent ions (Ca2+ and Mg2+) via spectra analysis. Both terrigenous and autochthonous DOM performed as critical DBP precursors, and DBP formation patterns were tightly correlated to organic matter chemical variations. DBP formation was significantly higher in drought period compared to that in wet period (P < 0.05). Particularly, trichloromethane (TCM) and dichloroacetonitrile (DCAN) showed distinct formation patterns compared to the scenarios in non-karst water. For DOM fractions, hydrophobic components showed higher DBP formation compared to hydrophilic counterparts, hydrophilic neutral enriched more reactive organic nitrogen for N-DBPs production. It was preferable to enrich humic-like substances after Ca2+ and Mg2+complexation in Chlorella sp. AOM, TCM formation increased whereas DCAN production remained unchanged in the presence of divalent ions. This study innovatively provided a linkage between chemical characteristics of DOM and understanding of DBP formation in karst surface water.