Due to different implementations in the execution of the teaching profession in Turkey, the aim of the study was to compare the Technological Pedagogical Content Knowledge (TPACK) self-efficacy levels of prospective Biology teachers' Faculty of Education (FoE) and the Faculty of Science (FoS) between 2013-2014 and 2017-2018 academic years. The research was carried out using a screening model, one of the quantitative research techniques. The participants of this study were selected via convenience sampling method, and consisted of a total of 342 volunteer prospective teachers, 138 from the faculty of education and 204 from the faculty of science. The data collection tool was the TPACK self-efficacy scale. The data were analyzed using independent sample T-test and one-way ANOVA, separately in five academic years. The results revealed that the TPACK self-efficacy scores of prospective Biology teachers of the faculty of education was higher than those who graduated from the faculty of science. In addition, when compared over the five academic years, it was also found that there was a significant difference among them in favor of the prospective Biology teachers of the faculty of education.