The mastery of Technological Pedagogical Content Knowledge (TPACK) is very important for teachers in order to achieve qualified learning. Analyzing the mastery of TPACK by teachers in Indonesia seems to be imperative to provide baseline data to Indonesian government. This study aimed to examine the mastery of TPACK, in particular among biology teachers in terms of their teacher certification status, educational level, and academic background. This research involved 68 biology teachers obtained through proportional random sampling in five districts of Yogyakarta Special Region, Indonesia. The data collection was conducted through a multiple-choice test consisting of 33 items. The results showed that although the teacher’s mastery of TPACK was categorized as fair (60.13), it does not meet the work performance standards as a good teacher in Indonesia which has a minimum score of 76.00. Using Mann Whitney U and Kruskal Wallis Tests (p< 0.05), the evidence revealed that the mastery of TPACK among biology teachers is likely influenced by the academic background, educational level, and teacher certification status.