The aim of this in vivo study was to test the diagnostic accuracy of a pen-type laser fluorescence (LFpen) device in detecting approximal caries lesions, in posterior permanent teeth, at the cavitation and non-cavitation thresholds, and compare it with that of digital bitewing radiography. Thirty patients (aged 18–37), who attended the Faculty of Dentistry at Damascus University for a dental examination, were consecutively screened. Ninety approximal surfaces of posterior permanent teeth without frank cavitations, enamel hypoplasia or restorations were selected and examined using the LFpen (DIAGNOdent pen) and digital bitewing radiography. The reference standard was the visual-tactile inspection, after performing temporary tooth separation, using orthodontic rubber rings, placed for 7 days. The status of included approximal surfaces was recorded as intact/sound, with white/brown spots or cavitated. One trained examiner performed all examinations. There were statistically significant differences in LFpen readings between the three types of approximal surface status (P < 0.001). The optimal cut-off values for detecting approximal caries lesions in posterior permanent teeth were >16 and 8 at the cavitation and non-cavitation thresholds respectively. The sensitivity, specificity and accuracy (measured by the area under the receiver-operating characteristic curve) were 100, 85 and 95 and 92, 90 and 95% at the cavitation and non-cavitation thresholds respectively. The intra-class correlation coefficient for intra-examiner reliability was 0.95. The diagnostic accuracy of the LFpen was significantly higher than that of digital bitewing radiography (P < 0.001). The LFpen’s diagnostic performance was accurate and significantly better than digital bitewing radiography in detecting approximal caries lesions, in posterior permanent teeth.Electronic supplementary materialThe online version of this article (doi:10.1007/s10103-017-2157-2) contains supplementary material, which is available to authorized users.