Cartoons appear to be an important teaching strategy, with numerous benefits in science classrooms. However, they have received little attention in Thai education as well as in biology concepts. The purpose of this research is to compare the conceptual understanding of biology of 30 freshmen pre-service teachers studying at the Faculty of Education, Rajabhat University, who registered a bioscience course in the second semester of the 2020 academic year by using four 5E lesson plans with biology concept cartoons:1. Cell transportation (4 h/week), 2. genetics (4 h/week), and 3. Ecosystems (4 h per week), 4. Evolution (4 h a week): 16 items of the two-tier multiple-choice test. The results showed that the biology concept cartoon lesson had a significant difference in academic achievement score, which was significantly higher than the pre-test scores (p < .05).