Nowadays, dental caries is one of the most common oral health problems, affecting most individuals. It has been found that, by remineralizing enamel at an early stage in the formation of enamel caries, teeth can be effectively protected from dental caries. In this work, a peptide with eight repetitive sequences of aspartate-serine-serine (8DSS) is applied as the bio-mineralizer in an in-vivo rat enamel caries model. Nondestructive quantitative light-induced fluorescence-digital (QLF-D) imaging and micro-computed tomography (micro-CT) are used to evaluate the remineralization of enamel carious lesions by measuring the total fluorescence radiance loss of the molar area (Δ QTotal), acquired using QLF-D imaging, and the mineral density and residual molar enamel volume, acquired using micro-CT. Correlations are explored between Δ QTotal and mineral density (strong correlation, r = 0.8000, p < 0.001) and Δ QTotal and residual molar enamel volume (moderate correlation, r = 0.6375, p < 0.001). Our results demonstrate that 8DSS is a promising in-vivo remineralization agent that exhibits comparable effects to NaF ( p < 0.05), which has been verified using the classical Keyes method. Moreover, the nondestructive QLF-D and micro-CT methods can be combined to quantify the remineralization of enamel carious lesions three-dimensionally in vivo, making them broadly applicable in quantifying hard tissues.