Little is known about how the chemical composition of enamel affects the optical properties of teeth, but advances in technology allow this to be studied using white-light and fluorescent images. This study aimed to identify the variation in enamel chemical composition that may affect tooth optical properties, such as tooth color and autofluorescence. Sixty-one specimens of extracted human molars were prepared. Raman spectrometry was used to assess chemical composition of enamel, and tooth color, and autofluorescence from enamel were evaluated by quantitative light-induced fluorescence (QLF) images. Pearson correlation and multiple linear regression analyses were used. Enamel fluorescence was related to enamel composition rather than tooth color. The b* value from the fluorescence image had a moderate correlation with crystallinity (full-width at half-maximum: r = −0.433, p < 0.001) and laser-induced fluorescence intensity (r = 0.450, p < 0.001) from Raman spectroscopy. In multiple linear regression analysis, the chemical composition of the tooth had a significant effect on the b* value from the fluorescent image (R2 = 0.433, p < 0.001). In contrast, tooth color values (L*, a*, and b*) were not correlated with chemical composition. The present study revealed that enamel autofluorescence in QLF was related to chemical composition of the enamel, particularly the inorganic‒organic interface. While enamel chemical composition can be detected only in a laboratory environment, enamel fluorescence by QLF may enable estimation in a dental clinic, which has implications for the field of tooth bleaching or esthetic restorative materials.