This study aimed to apply fluoride formulations to enamel with cold atmospheric plasma (CAP) and analyze the fluoride uptake, retention, and acid resistance quantitatively. Human enamel specimens were divided randomly into 2 groups: group APF1, 1.23% acidulated phosphate fluoride (APF) gel; group APF2, 1.23% APF gel with CAP. Fluoride and CAP were applied to the samples 4 times at 1-wk intervals. The specimens were also stored in artificial saliva for 4 wk to evaluate the retention of fluoride. The fluoride content on the fluoride-treated enamel was measured by an electron probe microanalyzer. To detect the resistance to demineralization, the calcium-to-phosphate ratio of the enamel samples was measured after the application of APF gel with or without CAP, followed by soaking in the demineralization solution. In groups APF1 and APF2, the amount of fluoride detected increased depending on the application frequency, and more fluoride was detected in group APF2 than in group APF1. In the experiment examining the maintenance effect, fluoride was not detected in group APF1, whereas fluoride was detected in group APF2 up to the fourth week. As for the resistance to demineralization, the calcium-to-phosphate ratio of the enamel treated with APF and CAP was higher than that treated with APF alone, and it increased with the frequency of treatment. This study suggests that the combination treatment of CAP and fluoride improves retention of fluoride on the enamel and resistance to demineralization when compared with treatment with fluoride alone.