The hydrogeochemical characteristics of aquifers in coal-bearing strata can provide an important foundation for understanding the evolution of the aquifers and recognizing the formation process and occurrence of biogenic gas. By analyzing the hydrogeochemical characteristics of water produced from the Cenozoic, the limestone, and the coal strata aquifers and the coalbed gas wells, this study focused on water-rock interactions and methanogenesis in the formation water of the Suzhou mining area, Huaibei coalfield, China. Tectonically, this is one of the most complex coalfields in China. The dissolution of evaporites, silicates, and carbonates controlled the chemical composition of the formation water. The Cenozoic aquifers have a relatively stable water quality type and balanced ionic composition, but more complex water-rock interactions occur in the coal strata and the limestone aquifers. Extensive methanogenic activity and methanogenesis could be recognized by the meteoric water recharge, positive δ¹³C signatures for dissolved inorganic carbon(DIC; 15.4‰–26.2‰), lower sulfate concentrations (<2 mM), and higher levels of dissolved methane and DIC. The generation and enrichment of secondary biogenic gas in the coal seams was after the microbes’ inoculation along with the freshwater infilling the fracture network.