Dentinal hypersensitivity (DH) is a common clinical condition usually associated with exposed dentinal surfaces. The aim of this study was to study the effect of sodium fluoride varnish, Gluma, and Er,Cr:YSGG laser, in the dentin hypersensitivity treatment. One hundred sixty-five teeth with dentin hypersensitivity in 55 patients were involved in this study. Teeth are divided into five groups based on the received treatment (n = 33): G group: Gluma; F group: sodium fluoride varnish (5%); L group: Er,Cr:YSGG laser (wavelength 2780 nm, frequency 20 Hz, power 0.25 W, energy density 44.3 J/cm 2 , and pulse width of 150 µs at distance of 1 mm for 30 s) which was followed by Er,Cr:YSGG laser; GL group: Gluma + laser; VL group: both sodium fluoride varnish and Gluma, which are common treatments for hypersensitivity, were selected as control groups. The treatment was performed in one session, and the sensitivity to air spray conditioning was recorded after the treatment, at 15 min, 1 week, 1 month, and 6 months as the VAS. Statistical analysis was performed using SPSS Ver. 21 software. One-way ANOVA was used to compare the VAS between all treatment groups at each time-point. One-way repeated measurements ANOVA (RM-ANOVA) and two-way-repeated measurements ANOVA (RM-ANOVA) were used to compare the hypersensitivity of each group and sensitivity of all treatment groups, respectively. Tukey post hoc test was used to compare the groups pairwise. The hypersensitivity between different groups at before and 15 min after the treatment was not significantly different (P = 0.063). The hypersensitivity of all studied groups was decreased after the treatment. The Er,Cr:YSGG laser, alone or in combination with Gluma, in 1 week, 1 month, and 6 month follow-ups, had significantly reduced the hypersensitivity instead of sodium fluoride varnish. All treatments significantly reduced the dentin hypersensitivity up to 6 months. Er,Cr:YSGG laser alone or in combination with Gluma was more effective than sodium fluoride varnish; however, it was not significantly different from other treatments. In a 6-month follow-up of dentine hypersensitivity treatment, Gluma had a significantly higher effect than sodium fluoride. Trial registration: IRCT20190422043343N1. Registered 19 July 2019. Keywords Dentinal tubule • Desensitizer agent • VAS score Abbreviations DH Dentinal hypersensitivity RM-ANOVA Repeated measurements ANOVA SRP Scaling and root planing VAS Visual analog scale SP Seal and protect * Mahsa Forouzande