The aim of this study was to evaluate the applicability of laser-induced autofluorescence (LIAF) spectroscopy to detect and quantify dental plaque. LIAF spectra were recorded in situ from dental plaque (0–3 grades of plaque index) in 300 patients with 404 nm diode laser excitation. The fluorescence intensity ratio of the emission peaks was calculated from the LIAF spectral data following which their scatter plots were drawn and the area under the receiver operating characteristics were calculated. The LIAF spectrum of clinically invisible grade-1 plaque showed a prominent emission peak at 510 nm with a satellite peak around 630 nm in contrast to grade 0 that has a single peak around 500 nm. The fluorescence intensity ratio (F510/F630) has a decreasing trend with increase in plaque grade and the ratio values show statistically significant differences (p<0.01) between different grades. An overall sensitivity and specificity of 100% each was achieved for discrimination between grade-0 and grade-1 plaque. The clinical significance of this study is that the diagnostic algorithm developed based on fluorescence spectral intensity ratio (F510/F630) would be useful to precisely identify minute amounts of plaque without the need for disclosing solutions and to convince patients of the need for proper oral hygiene and homecare practices.