Nucleation of crystals from solution is fundamental to many natural and industrial processes. In this work, the molecular mechanism of conformational polymorphism nucleation and the links between the molecular conformation in solutions and in crystals were investigated in detail by using 5-nitrofurazone as the model compound. Different polymorphs were prepared, and the conformations in solutions obtained by dissolving different polymorphs were analysed and compared. The solutions of 5-nitrofurazone were proven to contain multiple conformers through quantum chemical computation, Raman spectra analysis, 2D nuclear Overhauser effect spectroscopy spectra analysis and molecular dynamics simulation. The conformational evolution and desolvation path was illustrated according to the ¹H NMR spectra of solutions with different concentrations. Finally, based on all the above analysis, the molecular conformational evolution path during nucleation of 5-nitrofurazone was illustrated. The results presented in this work shed a new light on the molecular mechanism of conformational polymorphism nucleation in solution.