Oral health plays a crucial role in children’s growth and development, yet dental caries remains a common problem among preschoolers. Data collected at RA Al-Ikhlas Tuban revealed a high def-t score of 14.3%, indicating that preschoolers have limited knowledge of oral health. This is influenced by a lack of early education and the use of media that is not suitable for preschoolers. One example of a medium that can help improve children’s knowledge is the pop-up book; this medium is used because it features attractive, interactive three-dimensional visuals that can capture the attention of preschoolers. The purpose of this study is to determine the effect of using pop-up books on oral health knowledge among preschoolers at RA Al-Ikhlas Tuban. The method used in this study was a quantitative method with a pre-experimental design employing a one-group pre-test and post-test design. The independent variable of the study was the use of pop-up books as a medium for oral health education, and the dependent variable was the level of knowledge of preschool children regarding oral health. Sampling in this study used the total sampling technique, with a total sample size of 43 respondents. Data were collected using questionnaires administered before and after oral health education using pop-up books. Data were analyzed using the Wilcoxon Signed-Rank Test. The results of the study indicate that the use of pop-up books has a positive effect on improving knowledge of oral health. The average knowledge score after the intervention was higher than before the intervention, with a significant p-value of 0.00 (p < 0.05). Pop-up books were found to be effective in helping preschool children understand oral health material because the information is presented in a more visual, engaging, and easy-to-understand manner. Based on the research results, it was concluded that the use of pop-up books has an effect on improving oral health knowledge among preschool children at RA Al-Ikhlas Tuban.