Enamel demineralization is a common complication of orthodontic treatment. Studies related to enamel demineralization are usually conducted in the laboratory using in vitro models. Detection technology relies on large instruments, which are not conducive to its popularization and promotion. We designed a fluorescence sensing system using a complex formed by 3,4,9,10-tetra-(4-trimethylaminohexyloxy-carbonyl)-perylene (PDI-HTMA) and casein phospho-peptides (CPPs) that can rapidly detect calcium ions in enamel. In this study, we investigated the feasibility of using PDI-HTMA/CPPs as probes for detecting enamel demineralization. We used Ca2+ from enamel demineralization as the detection target for the fluorescent probe and verified its stability, specificity, and linear range. This method exhibits strong linear relationships in the Ca2+ concentration range of 30–500 μM, with a limit of detection of 3.89 μM. The PDI-HTMA/CPPs were applied to human enamel specimens. We compared the detection results with those of existing enamel demineralization evaluation methods, and the results showed a trend consistent with that of ICDAS grading and laser fluorescence caries detection. This fluorescent probe assay is expected to be applied for the early detection of enamel demineralization, which has strong application advantages. To the best of our knowledge, this is the first fluorescent probe assay applied for the in vitro detection of enamel demineralization.