Chemical exchange saturation transfer (CEST) sensitively detects molecular alterations in the brain, such as relayed nuclear Overhauser effect (rNOE) CEST contrast at -3.5 ppm representing aliphatic protons in both lipids and proteins, and CEST contrast at 3.5 ppm correlating with amide proton in proteins. Myelin is rich in lipids and proteins, and therefore CEST can be explored as a biomarker for myelin pathology, which could contribute to the diagnosis and prognosis of multiple sclerosis (MS). In the current study, we investigate the specificity of aliphatic rNOE and the amide pool in myelin detection using the cuprizone (CPZ) mouse model, which recapitulates the demyelination and remyelination of MS. In this study, preclinical 3T MRI was performed in 19 male C57BL/6 mice. Mice in the normal control (NC) group (n = 9) were fed a normal diet for the whole course, while mice in the CPZ group (n = 10) were fed with CPZ for 10 weeks, followed by 4 weeks with a normal diet. The CEST contrast of rNOE (-3.5 ppm) and amide (3.5 ppm) in brain regions of the corpus callosum (CC) and the caudate putamen were compared. Statistical differences between the groups were calculated using two-way ANOVA. We observed significantly decreased rNOE (NC: 4.85% ± 0.09%/s vs. CPZ: 3.88% ± 0.18%/s, p = 0.007) and amide pool (NC: 3.20% ± 0.10%/s vs. CPZ: 2.46% ± 0.16%/s, p = 0.02) in the CC after 8 weeks on CPZ diet (p < 0.05). Moreover, the rNOE in the CPZ group recovered to a level comparable with the NC group at week 14 (p = 0.39), while amide remained at a level as low as that for the NC group (p = 0.051). Significant rNOE and amide changes, validated by immunohistochemistry results for demyelination and remyelination, demonstrate the huge potential of CEST for revealing myelin pathology, which has implications for MS identification at the clinical field strength of 3T.