Purpose: The aim of this study was to evaluate the validity of swept-source optical coherence tomography (SS-OCT) for detecting sealant microleakage. Materials and Methods: A total of 31 extracted sound human molar, assigned as suitable for sealant application, were chosen and divided into two groups: (1) no microleakage group and (2) microleakage group by applying sealant using the different methods. All specimens were immersed in 1% methylene blue for 24 h and sectioned to confirm the absence or presence of sealant microleakage as a gold standard method. Subsequently, all the sectioned specimens were digitally photographed using a microscope with a magnification of 50. Presence of microleakage was evaluated on a 2-point rating scale. The association of histological method with conventional methods (visual and tactile assessment) and SS-OCT was assessed using a chi-squared test. The intra- and inter-examiner reliability was calculated using Cohen’s Kappa. Results: The SS-OCT showed a sensitivity of 0.73 and a specificity of 1.00, while visual and tactile assessment showed a sensitivity of 0.67 and a specificity of 0.86. The inter-examiner reliability of SS-OCT was 0.79, whereas that of the visual and tactile assessment was 0.53. Conclusions: SS-OCT can be used to non-invasively detect sealant microleakage and to monitor pit and fissure sealant in the clinics.