Background <p>In the context of the rapid development of artificial intelligence (AI) technology, questioning has become an increasingly active and critical component of effective collaboration with AI. Also, the ability to apply knowledge from various disciplines to analyze and address problems is essential for enhanced learning. Despite its importance, studies on training students in questioning and knowledge integration within course teaching are lacking.</p> Purpose <p>This study aimed to explore the effects of an innovative teaching approach based on the Bloom-based “Questioning-Training of Comprehensive Knowledge Application (Q-TOCKA)” method. This approach integrates a teacher-AI-student interaction model, promoting deeper engagement in the learning process.</p> Methods <p>The study included two groups of students: a control class group and an experimental class group. In the context of four problem-based learning (PBL) assignments during the course of Pharmaceutical Botany and Pharmacognosy, the experimental class was subjected to Bloom-based Q-TOCKA of progressive exercises, paired with the teacher-AI-student in-depth interaction and communication model. Conversely, the control class followed the traditional teacher-student dichotomous communication method. The students’ questioning and knowledge application scores in both classes were analyzed using R statistical software, interaction model regression analysis, and prediction model Holt linear trend method analysis. Additionally, the data from a course questionnaire survey for students in the experimental class were statistically collected.</p> Results <p>Statistical analysis using R revealed significantly higher scores of the experimental class in questioning and comprehensive application of knowledge across the two to four post-course PBL assignments (<i>P</i> &lt; 0.05) compared with the control class. Pearson correlation analysis indicated a linear correlation between questioning scores and comprehensive knowledge application scores. Furthermore, the regression analysis of the interaction model suggested a synergistic relationship between questioning ability and comprehensive knowledge application. Holt linear trend method indicated that the positive impact of the new teaching method on students’ questioning and knowledge application abilities became more pronounced with an increase in the number of training sessions. The questionnaire survey results showed that more than 90% of the experimental-class students expressed a favorable attitude toward the new teaching approach.</p> Conclusions <p>The innovative teaching method significantly enhanced students’ questioning abilities and their capacity for comprehensive knowledge application. The positive feedback from students indicates that this teaching approach holds promise for broader application and promotion across various courses.</p>