Dentinal hypersensitivity is characterized by short, sharp pain from exposed dentine that occurs in response to external stimuli such as cold, heat, osmotic, tactile or chemicals, and cannot be explained by any other form of dental defect or pathology. Laser therapy has become a commonly used intervention and might be effective for dentinal hypersensitivity. To assess the effects of in-office employed lasers versus placebo laser, placebo agents or no treatment for relieving pain of dentinal hypersensitivity. Cochrane Oral Health's Information Specialist searched the following databases: Cochrane Oral Health's Trials Register (to 20 October 2020), the Cochrane Central Register of Controlled Trials (CENTRAL) (the Cochrane Library 2020, Issue 9), MEDLINE Ovid (1946 to 20 October 2020), Embase Ovid (1980 to 20 October 2020), CINAHL EBSCO (Cumulative Index to Nursing and Allied Health Literature; 1937 to 20 October 2020), and LILACS BIREME Virtual Health Library (Latin American and Caribbean Health Science Information database; from 1982 to 20 October 2020). Conference proceedings were searched via the ISI Web of Science and ZETOC, and OpenGrey was searched for grey literature. The US National Institutes of Health Ongoing Trials Register (ClinicalTrials.gov) and the World Health Organization International Clinical Trials Registry Platform were searched for ongoing trials. No restrictions were placed on the language or date of publication when searching the electronic databases. Randomized controlled trials (RCTs) in which in-office lasers were compared to placebo or no treatment on patients aged above 12 years with tooth hypersensitivity. Two review authors independently and in duplicate screened the search results, extracted data, and assessed the risk of bias of the included studies. Disagreement was resolved by discussion. For continuous outcomes, we used mean differences (MD) and 95% confidence intervals (CI). We conducted meta-analyses only with studies of similar comparisons reporting the same outcome measures. We assessed the overall certainty of the evidence using GRADE. We included a total of 23 studies with 936 participants and 2296 teeth. We assessed five studies at overall low risk of bias, 13 at unclear, and five at high risk of bias. 17 studies contributed data to the meta-analyses. We divided the studies into six subgroups based on the type of laser and the primary outcome measure. We assessed the change in intensity of pain using quantitative pain scale (visual analogue scale (VAS) of 0 to 10 (no pain to worst possible pain)) when tested through air blast and tactile stimuli in three categories of short (0 to 24 hours), medium (more than 24 hours to 2 months), and long term (more than 2 months). Results demonstrated that compared to placebo or no treatment the application of all types of lasers combined may reduce pain intensity when tested through air blast stimuli at short term (MD -2.24, 95% CI -3.55 to -0.93; P = 0.0008; 13 studies, 978 teeth; low-certainty evidence), medium term (MD -2.46, 95% CI -3.57 to -1.35; P < 0.0001; 11 studies, 1007 teeth; very low-certainty evidence), and long term (MD -2.60, 95% CI -4.47 to -0.73; P = 0.006; 5 studies, 564 teeth; very low-certainty evidence). Similarly, compared to placebo or no treatment the application of all types of lasers combined may reduce pain intensity when tested through tactile stimuli at short term (MD -0.67, 95% CI -1.31 to -0.03; P = 0.04; 8 studies, 506 teeth; low-certainty evidence) and medium term (MD -1.73, 95% CI -3.17 to -0.30; P = 0.02; 9 studies, 591 teeth; very low-certainty evidence). However, there was insufficient evidence of a difference in pain intensity for all types of lasers when tested through tactile stimuli in the long term (MD -3.52, 95% CI -10.37 to 3.33; P = 0.31; 2 studies, 184 teeth; very low-certainty evidence). Most included studies assessed adverse events and reported that no obvious adverse events were observed during the trials. No studies investigated the impact of laser treatment on participants' quality of life. Limited and uncertain evidence from meta-analyses suggests that the application of laser overall may improve pain intensity when tested through air blast or tactile stimuli at short, medium, or long term when compared to placebo/no treatment. Overall, laser therapy appears to be safe. Future studies including well-designed double-blinded RCTs are necessary to further investigate the clinical efficacy of lasers as well as their cost-effectiveness.