TPACK questionnaires have been extensively developed, but measurements considering biological content insights still need to be explored. This study aimed to develop and validate TPACK questionnaires by assessing core competencies, core content, and interdisciplinary biology knowledge for preservice biology teachers. We performed development and validation through focus group discussions with biological education experts, and survey studies. The survey was conducted in three phases with a total number of 732 preservice biology teachers. In phase 1, descriptive analysis was calculated based on 232 participants. In Phase 2, Exploratory Factor Analysis (EFA) was calculated based on 250 participants. In phase 3, Confirmatory Factor Analysis (CFA) was calculated to validate and confirm the final dimension of the instrument recently developed based on 250 participants. This series of analyses resulted in 44 items with eight dimensions: Technological Pedagogical Biological Content Knowledge of Biology (TPACKB) comprises 12 items; Technological Knowledge (TK) of biology, Biological Knowledge (BK), and Technological Pedagogical Knowledge (TPK) of the biology consist of 6 items respectively; Technological Biological Knowledge (TBK) comprises of 5 items; Pedagogical Knowledge (PK) of biology, Pedagogical Biological Knowledge (PBK), and Biological Context Knowledge (BCxK) comprise of 3 items respectively. Our study recommended a TPACK questionnaire to assess preservice biology teachers' holistically interdisciplinary understanding, core content, and core competencies as necessary steps to empower biological resolution for their students.