This study aimed to investigate and compare fluoride levels in saliva and plaque between the MU caries preventive program and a standard program. A randomized controlled trial was conducted on 77 preschool children from five daycare centers in Pathum Thani, Thailand. Children were randomly arranged into 2 groups: 1) a control group was provided a standard program including oral examination, oral hygiene instruction, diet advice and a fluoride varnish application; 2) a treatment group was provided the MU caries preventive program, which added extra interventions, including Interim Therapeutic Restoration (ITR) and sealant on posterior teeth with glass-ionomer cement. Plaque and saliva samples were collected before and after the program implementation at 24 hours, 1 week, 1 and 3 months, respectively. Salivary fluoride level was measured by a fluoride electrode, while plaque fluoride level was analysed by micro-diffusion technique and using a fluoride electrode (Model 96-09 Orion). The difference of plaque and salivary fluoride levels between the two groups was analyzed by Repeated ANOVA and Mann-Whitney U test, respectively. The treatment group showed a significantly higher plaque fluoride level than the control group at 24 hours (p<0.001), 1 week (p=0.018), and 1 month. (p=0.022). However, no significant difference was observed between the two groups at 3 months (p=0.228). The salivary fluoride levels showed the same tendency. The treatment group showed significantly higher salivary fluoride levels than the control group at 24 hours (p<0.001), 1 week (p<0.001), and 1 month (p=0.028). However, no significant difference was observed between the two groups at 3 months (p=0.055). This study was concluded that the plaque and salivary fluoride levels of children in MU caries preventive program were significantly higher when compared with the standard program at 24 hours, 1 week and 1 month.