Values of the enthalpy of dilution were measured for l-prolinol in pure water and N,N-dimethylformamide (DMF) aqueous solutions with various mass fractions of DMF at T=298.15K using a flow-mixing microcalorimeter. A pseudo phase equilibrium model was proposed to simplify the complex aggregation equilibrium and interpret the abnormality in the dilution enthalpy, which together with the McMillan–Mayer approach was used to fit the experimental data to obtain the enthalpic pairwise interaction coefficients and the molar aggregation enthalpies of l-prolinol in DMF aqueous solutions. The results are discussed in terms of the hydrophobic interaction and the interactions between the solvated solutes.