The principal aim of this in vitro study was to assess the validity and reproducibility of the detection of occlusal caries using laser fluorescence (LF), prior to and following the placement of a clear fissure sealant. It also aimed to compare the manufacturer's standard cut-off recommendations with those published for in vitro studies and to compare the validity and reproducibility of LF with clinical visual examination (CVE) for the detection of occlusal caries under sealants. Three clinicians independently examined visually and with LF 37 extracted teeth (25 molars, 12 premolars), with a range of clinical caries from apparently sound to cavitated dentinal caries. Examinations were conducted under dental surgery conditions. Subsequently, the teeth were serially sectioned to provide the validating criterion. Following placement of the sealant, the specificity generally increased but there was an associated loss of sensitivity, at both the D1(enamel and dentine) and D3 (dentine) diagnostic thresholds. The LF readings were significantly lower after placement of the sealant (p < 0.05). The manufacturer's recommended cut-offs appear to be the most appropriate to use. The CVE had superior validity and reproducibility when compared to LF. Overall, the placement of a clear sealant does influence the detection of caries by LF but does not prevent the detection of caries by this method.