G-quadruplexes are known to be potential targets for therapeutic intervention, thus resulting in development of various quadruplex interacting ligands. However, until now, no systemic study has been performed to understand molecular recognition of quadruplex in the presence of molecular crowding agents mimicking cellular conditions. The stability and molecular recognition of quadruplex can be influenced by loop length. Herein, we attempted to study the interaction of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine (TMPyP4), a well-known G-quadruplex binding ligand with various DNA quadruplexes differing in total loop length and loop arrangement in both the absence and presence of molecular crowding agents. Results obtained from CD studies revealed that longer loops favor mixed and antiparallel conformation in both the absence and presence of 30% ethylene glycol. UV thermal melting studies revealed that the stability and formation of quadruplex increases in the presence of 30% ethylene glycol. Moreover, the binding of TMPyP4 molecule to both of the binding sites in different quadruplexes with total loop length varying from 3 to 9 remains unchanged in both the absence and presence of 30% ethylene glycol. The binding affinity (K-a) of TMPyP4 was found to be decreased approximately by 1 order for the quadruplex sequences with total loop length varying from 11 to 15 in the presence of molecular crowding agents.