Purpose: The purpose of this study was to evaluate the effect of fluoride varnish in preventing dental caries of permanent first molars (PFMs). Methods: The study was designed to be a stratified-cluster randomized controlled trial with classes used as the unit of randomization. Classes stratified by district were followed for 36 months. All eligible children of the selected classes were included in the trial. The children in the test group were applied fluoride varnish biannually. The outcomes were measured at the individual level. Results: In total, 107 classes (51 in the test group, 56 in the control group) were recruited for the trial. Among the 5,397 total subjects, 5,005 and 4,596 children completed the 24-month and 36-month course, respectively. There were no group differences at baseline (P>0.05). The mean decayed and filled surfaces scores of the test group were significantly lower than those of the control group at the 36-month follow-up (P<0.05). The caries processing speed of PFMs increased from 24 months to 36 months; however, group differences were not significant (β equals 0.01; P>0.05). Conclusions: Biannual application of fluoride varnish can effectively prevent dental caries of six- to seven-year-old children. Nevertheless, the use of fluoride varnish with additional treatments (such as pit and fissure sealants) should be considered for optimized benefit after 24 months.