Objectives This randomized controlled trial aimed to evaluate different protocols for dentin hypersensitivity treatment with low-power lasers and desensitizing agents, and the association between low-power lasers and desensitizing agents. Materials and methods Fifty-four patients (303 teeth) were randomly allocated to three groups: G1, 3% nitrate potassium gel, UltraEZ ( n = 17); G2, photobiomodulation therapy (PBM) with a low-level infrared laser ( n = 17), 100 mW, spot size of 0.028 cm 2 , and dose of 1 J per point; and G3, nitrate potassium + PBM ( n = 20). Treatments were applied to the buccal cervical region at intervals of 72 h, and all protocols were performed in three sessions. The patients’ response to evaporative stimuli was rated using the visual analog scale (VAS). Re-evaluations were performed immediately after each application and 1 week, 1 month, and 3 months after treatment. A two-way repeated measures test and Tukey’s post hoc test were used for multiple comparisons ( α = 5%). Results There was a reduction in pain levels at the end of treatment in all groups. There were no significant differences in VAS score changes between the groups immediately after treatment and after the third month, compared to the baseline ( p > 0.05). Conclusion Under the limitations of this in vivo study, the proposed three-session protocol was effective in reducing dentin hypersensitivity after 3 months, regardless of the desensitization mechanism used. Conservative and long-term protocols are interesting for the control of pain caused by dentin hypersensitivity. Clinical relevance The increase in cervical dentin hypersensitivity prevalence warrants easy-to-apply and long-lasting desensitizing protocols for pain control.