Oxidative polymerization of phenols using horseradish peroxidase (HRP) in aqueous organic solvents (1,4-dioxane-water and methanol-water), which is an attractive pathway for synthesis of a new class of polyphenols without use of toxic formaldehyde, was remarkably influenced by the water content. The present study is focused on the self-association of in-cresol in such a solvent to understand the effect of solvent on its polymerization. The change of the molecular weight of the polymer with varying the water content was found to be related to the molecular clustering in the solution, observed through the difference UV absorption spectroscopy and the mass spectrometry for clusters. In mixed solvents of 1,4-dioxane and aqueous 0.1 M phosphate buffer (pH 7), number-average molecular weight had a maximum at the aqueous buffer content of 50 vol %, whereas in methanol-aqueous buffer mixtures, the molecular weight had a small maximum at 20 vol % aqueous buffer content and gradually decreased with increase of the aqueous buffer content. As for the in-cresol clustering, the effect of the water content was also in significant contrast between the above aqueous organic solvent systems. The in-cresol clusters were not formed in 1,4-dioxane, but its clustering was remarkably promoted by an increase of the water content. On the other hand, the in-cresol clusters were formed favorably in methanol and were readily hydrated with increase of the water content in the solution. These effects of the water content on the clustering are related to the enzymatic polymerization. The polymer would be obtained in high yields when the m-cresol clusters were formed in the solution: however, the hydration of the in-cresol cluster would restrain the polymerization.