This in vitro study aimed to evaluate the feasibility of quantitative light-induced fluorescence (QLF) technology for detecting the presence and severity of microleakage of pit and fissure sealants. The areas of interest (AOIs) were 160 pits and fissures of 40 extracted permanent teeth. Fluorescent images were acquired using a QLF device, and the maximum fluorescence loss ΔFmax of each AOI was analyzed. After staining and cross-sectioning of the teeth, histological dye penetration was scored on a scale of 0 to 3. The relationship between ΔFmax and microleakage depth was analyzed, and the areas under the curve (AUCs) were calculated. The │ΔFmax│ increased as microleakage depth increased. The ΔFmax values of microleakage areas showed a strong significant correlation with the histological scores of dye penetration (r = − 0.72, P = 0.001). AUC analysis showed a high diagnostic accuracy for microleakage depth (AUC = 0.83–0.91). The highest AUC of 0.91 was found when differentiating the outer half microleakage of the sealant (histological score 0 vs. 1–3). QLF technology is effective in assessing the presence and severity of microleakage, suggesting its potential for noninvasive detection and monitoring of sealant microleakage in clinical settings.