Purpose Enamel demineralization can occur as a side effect during orthodontic treatment with fixed appliances and should be detected as early as possible. A new approach to assess demineralization is a system consisting of a photosensitive protein that binds to free calcium ions at the enamel surface. A camera is then used to visualize the bioluminescence spots. This in vitro study aimed to evaluate the ability of the bioluminescence technology to assess artificially demineralized enamel adjacent to various orthodontic brackets. Methods In all, 108 human enamel samples were allocated randomly to groups with different orthodontic bracket material: stainless steel, titanium, ceramic. Initial lesions were created adjacent to the brackets. The samples were assessed by bioluminescence before and after demineralization. Images were assessed for presence of bioluminescence spots (yes/no). To quantify the bioluminescence measurements, the images’ pixel values (P) were calculated within a defined area (F) adjacent to each bracket before and after demineralization. Quantitative light-induced fluorescence measurements (ΔF, ΔQ) were performed as the reference standard for demineralization. Results After demineralization, bioluminescence spots were visible (yes/no decision) in 87% of the samples. The pixel analysis of the bioluminescence spots showed significantly higher pixel values after demineralization compared to baseline (p < 0.0001). The bracket material had no influence on the bioluminescence measurements. All samples showed fluorescence loss with a median ΔF of −9.52% (±3.15) and a median ΔQ of −1.01% × mm2 (±3.34), respectively. Conclusion The bioluminescence technology is a promising tool to demonstrate demineralization adjacent to different orthodontic brackets in vitro.