CONTEXT: The main challenge of large-scale biofuel production is related to the extraction of its undesired impurities including glycerol, water, methanol, soap/catalyst, free fatty acids, glycerides, and others. There are many ways to remove glycerol, and herein, the one alternative is the extraction of glycerol from biodiesel by deep eutectic solvents. In this regard, the mixture of a choline chloride (ChCl) and urea, methyltriphenylphosphonium chloride (MTPPCl), and ethylene glycol (EGL), as a deep eutectic solvent (DES), is effective in removing glycerol from biofuel. METHODS: In this work, we have investigated the formation mechanism of ChCl and urea, and then MTPPCl and EGL, as a DES, and then extraction of glycerol from biofuel via DES implementing density functional theory (DFT) by Gaussian09 software, B3LYP basis set, and classical all-atom molecular dynamics (MD) simulations by Gromacs software, GROMOS force field. DFT approximation demonstrates that Cl ion plays an important binding role in the formation of complexes ChCl/urea-based DES + biofuel and in MTPPCl/EGL-based DES + biofuel. We have also considered the formation and change of hydrogen bonds upon the formation of these systems using the DFT method. Large HOMO-LUMO gaps in ChCl/urea-based DES + biofuel and in MTPPCl/urea-based DES + biofuel demonstrate the stability of the complexes. The results of MD work have stated that the chloride ion formed bonding with the choline/ethylene glycol EGL, while still weakly intermolecular interacting with the urea/methyltriphenylphosphonium in ChCl/urea- and MTPPCl/EGL-based DESs. Further results of MD simulations stated that the DESs had a higher intermolecular interaction with glycerol in comparison with biofuel, thereby favoring the extraction process of glycerol from model biofuel. HIGHLIGHTS: • Intermolecular interactions of choline chloride and urea, methyl triphenyl phosphonium chloride, and ethylene glycol-based DESs and their applications in the extraction of glycerol from biofuel studied by DFT calculations and classical all-atom molecular dynamics simulations. • Calculated outputs of DFT calculations and classical all-atom molecular dynamics simulations for DESs and their applications in the extraction of glycerol from biofuel were discussed in detail. • The molecular formation mechanism of choline and methyl triphenyl phosphonium-based DESs and their application in the extraction process of glycerol from biofuel were summarized.