The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim50 is a major receptor for transporting the precursor protein across the mitochondrial inner membrane in the TIM23 complex. However, the interaction of prequence peptide with Tim50 is not well-known. Here, we investigated in vitro the interaction of presequence peptide with the intermembrane space domain of Tim50 (Tim50(IMS)) by micro-Raman and fluorescence spectra. The fluorescence quenching revealed that the interaction between Tim50(IMS) and presequence peptide is mainly electrostatic interaction, and the distances between Tim50(IMS) and presequence peptides are estimated by fluorescence resonance energy transfer. Micro-Raman spectra showed that presequence peptides induce a more compact conformation of Tim50(IMS), and synchronous fluorescence showed that the tyrosine or tryptophan fluorescence quenching molar ratio of presequence peptide to Tim50(IMS) is less than 3.