Understanding molecular interactions and dynamics of proteins and DNA in a cell-like crowded environment is crucial for predicting their functions within the cell. Noncanonical G-quadruplex DNA (GqDNA) structures adopt various topologies that were shown to be strongly affected by molecular crowding. However, it is unknown how such crowding affects the solvation dynamics in GqDNA. Here, we study the effect of cosolvent (acetonitrile) crowding on ligand (DAPI) solvation dynamics within human telomeric antiparallel GqDNA through direct comparison of time-resolved fluorescence Stokes shift (TRFSS) experiments and molecular dynamics (MD) simulations results. We show that ligand binding affinity to GqDNA is drastically affected by acetonitrile (ACN). Solvation dynamics probed by DAPI in GqDNA groove show dispersed dynamics from ∼100 fs to 10 ns in the absence and presence of 20% and 40% (v/v) ACN. The nature of dynamics remain similar in buffer and 20% ACN, although in 40% ACN, distinct dynamics is observed in <100 ps. MD simulations performed on GqDNA/DAPI complex reveal preferential solvation of ligand by ACN, particularly in 40% ACN. Simulated solvation time-correlation functions calculated from MD trajectories compare very well to the overall solvation dynamics of DAPI in GqDNA, observed in experiments. Linear response decomposition of simulated solvation correlation functions unfolds the origin of dispersed dynamics, showing that the slower dynamics is dominated by DNA-motion in the presence of ACN (and also by the ACN dynamics at higher concentration). However, water-DNA coupled motion controls the slow dynamics in the absence of ACN. Our data, thus, unravel a detailed molecular picture showing that though ACN crowding affect ligand binding affinity to GqDNA significantly, the overall dispersed solvation dynamics in GqDNA remain similar in the absence and the presence of 20% ACN, albeit with a small effect on the dynamics in the presence of 40% ACN due to preferential solvation of ligand by ACN.