This study investigated biology teachers’ understanding and utilization of Technological Pedagogical Content Knowledge (TPACK) and Artificial Intelligence–integrated TPACK (AI-TPACK), with particular attention to subject major and prior experience with digital tools. Forty-two biology teachers and forty-two ethics teachers, selected to represent contrasting pedagogical contexts (inquiry-based vs. discourse-based) completed an online survey addressing their use of digital technologies, instructional integration of digital tools, and self-reported TPACK and AI-TPACK. Three key findings emerged. First, early-career biology teachers actively used digital tools for both instructional and administrative purposes, yet relied more on self-directed use of personal platforms than on formal institutional training to develop related knowledge. Second, biology teachers’ subject major did not significantly influence their AI-TPACK understanding or utilization, suggesting that personal digital practices may play a more decisive role than disciplinary background. Specifically, although the overall AI-TPACK score differed significantly between groups, the effect size was moderate, and among the sub-components, only AI-Ethic reached statistical significance, suggesting that the group difference was primarily driven by ethical awareness rather than by technical or pedagogical integration. Third, more frequent instructional use of digital tools was strongly associated with higher levels of both TPACK and AI-TPACK, underscoring the importance of accumulated, hands-on experience. These results highlight the need for biology teacher education programs that combine subject-sensitive instructional design with individualized, practice-based support for digital learning. AI-enhanced, personalized professional development may further strengthen biology teachers’ capacity to meaningfully integrate AI and digital tools into their instruction.