Z-DNA refers to the left-handed double-helix DNA that has attracted much attention because of its association with some specific biological functions. However, because of its low content and unstable conformation, Z-DNA is normally difficult to observe or identify. Up to now, there has been a lack of unified or standard analytical methods among diverse techniques for probing Z-DNA and its transformation conveniently. In this work, NaCl, MgCl2, and ethanol were utilized to induce d(GC)8from B-DNA to Z-DNA in vitro, and Fourier transform infrared (FTIR) spectroscopy was employed to monitor the transformation of Z-DNA under different induction conditions. The structural changes during the transformation process were carefully examined, and the DNA chirality alterations were validated by the circular dichroism (CD) measurements. The Z-DNA characteristic signals in the 1450 cm−1–900 cm−1region of the d(GC)8infrared (IR) spectrum were observed, which include the peaks at 1320 cm−1, 1125 cm−1and 925 cm−1, respectively. The intensity ratios of A1320/A970, A1125/A970, and A925/A970increased with Z-DNA content in the transition process. Furthermore, compared with the CD spectra, the IR spectra showed higher sensitivity to Z-DNA, providing more information about the molecular structure change of DNA. Therefore, this study has established a more reliable FTIR analytical approach to assess BZ DNA conformational changes in solutions, which may help the understanding of the Z-DNA transition mechanism and promote the study of Z-DNA functions in biological systems.