Background Fluoride varnish (FV) is an established technology for primary and secondary caries prevention. Objective The aim of this review was to evaluate the preventive effect of FV on development of white spot lesions (WSL) when regularly applied during orthodontic treatment with fixed appliances. Search methods We searched PubMed, Scopus and Google Scholar up to October 2022 using predetermined keywords. Selection criteria We included randomized controlled trials of a duration of minimum 12 months and at least quarterly FV applications. Data collection and analysis Based on abstracts, we retrieved full-text papers, extracted key outcome data, and assessed risk of bias. Primary outcome was prevalence of WSLs on subject level after debonding. We conducted a narrative synthesis and pooled comparable outcome data in a random effects model. Results We included seven studies covering 666 patients and assessed four publications with low or moderate risk of bias and three with high. The prevalence of WSLs at debonding varied between 12 and 55%. All studies presented results in favour for the FV intervention, one reached statistical significance on subject level. Five studies provided data for a meta-analysis. The pooled risk ratio was 0.64 [95% CI: 0.42, 0.98], indicating a statistically significant preventive effect. Certainty of evidence was graded as very low after reducing for risk of bias, inconsistency and imprecision. Limitations We pooled data on subject level and did not consider lesion severity on tooth level. Conclusions and implications Even if the certainty of evidence was very low, it was shown that FV can prevent development of WSL when regularly applied during orthodontic treatment. Larger investigations reporting a core outcome set are required to increase the certainty of evidence. Registration PROSPERO database (CRD42022370062).