Hydration plays a key role in the structure-specific stabilization of biomolecules such as nucleic acids. The hydration patterns of biased DNA sequences in the genome, such as GC-repetitive and AT-repetitive regions, are unique to their duplex grooves. As these regions are crucial for maintaining genomic homeostasis and preventing diseases such as cancer and neurodegenerative disorders, the effects of hydration on their stability and functions must be quantitatively analyzed in chemical environments that resemble intracellular conditions. In this study, we systematically investigated duplex formation of biased sequences in cell-like molecularly crowded environments to quantify the effects of groove hydration on their thermodynamics. The interaction of crowders with water molecules in the grooves was found to provide excess stabilization to biased DNAs than to unbiased DNAs, as estimated from the nearest-neighbor prediction model. These hydration effects are sequence-specific and depend on the cation type and cosolute size. Introduction of the "hydration parameters" into the nearest-neighbor model quantifying the effect of groove hydration remarkably enhanced the prediction accuracy for biased DNA stability in crowded environments. Hydration parameters can aid in elucidating the roles of biased sequences in cells such as cation-dependent quadruplex formation in cancer-related genes and regulation of replication initiation by intracellular crowding fluctuations. Additionally, these parameters can predict the free energy changes during the binding of protein to DNA grooves. Overall, our findings can help in realizing and predicting the functions of biased DNAs in cells controlled by variable chemical environments.