Structural change of lysozyme in the aqueous mixture of ethanol (ETH) has been investigated using emission, circular dichroism spectroscopy, free-energy molecular dynamics (MD) simulation, and contact map analysis methods. The emission and circular dichroism data of protein depict the nonmonotonic change suggesting that the structure as well as local environment near the Trp of lysozyme modifies differently for different compositions of the ETH-water mixture. The free-energy MD simulation shows that the change in the average size of lysozyme in the aqueous mixture of ETH also shows a nonmonotonic nature. The free-energy profile of lysozyme in the low concentration of ETH suggests that the distance between helices increases (chi(ETH) approximate to 0.07) first and decreases again (chi(ETH) approximate to 0.2) becoming almost similar to the native structure. Around chi(ETH) approximate to 0.5, the size of lysozyme increases significantly probably leading to the unfolding of the protein. With further increase of ETH (chi(ETH) approximate to 0.7), size of lysozyme decreases suggesting the refolding of the protein. Contact map as well as solvent organization analysis depicts that ETH gets preferentially solvated by the hydrophobic core of lysozyme which weaken the hydrophobic interactions of protein, resulting in the increase in its size. The aggregation of ETH dominated at the higher concentration of ETH (chi(ETH) approximate to 0.7); hence, the, weakening of hydrophobic core of protein by ETH is reduced, which probably lead to the refolding of lysozyme.