Scientific misconceptions are scientifically incorrect understandings or intuitions that negatively affect the learning and teaching process and are resistant to change. Teachers are one of the most common and important factors that cause students to develop misconceptions. The purpose of this study was to develop and implement a two-tier diagnostic test in order to identify prospective biology teachers' misconceptions and level of understandings of selected general biology concepts. The sample of the study (N = 296, (235 (79.4%) females, 62 (20.6%) males)) was consisted of senior biology education students and graduates of biology programs who were enrolled in pedagogical formation programs in five state universities in Turkey. Survey research model within the scope of the quantitative research is used. The final version of two-tier general biology concept diagnostic test consisted of twenty items, measuring understandings in nineteen selected subjects. The average item difficulty index of content information, which is the first tier of items, is 0.39, and the average item discrimination index is 0.34. The average item difficulty of the rationale, which is the second tier is 0.28 and the average item discrimination is 0.33 The Cronbach alpha reliability coefficient of the whole test is r = 0.68. Conceptual understandings of participants were classified based on their answers to test items as conceptual understanding, misconception, memorized knowledge, and inconsistent knowledge. The findings showed that prospective biology teachers have common misconceptions in many general biology subjects that are in the high school curriculum. At least 80% of the participants had misconceptions in circulatory system, protein synthesis and cell division subjects. The most common misconceptions that are identified in each subject of general biology are discussed in the light of the related literature and suggestions for prospective biology teachers, college biology educators and teacher educators are offered.