Teacher-related factors significantly impact students’ higher-order thinking. However, the mechanisms of how these factors specifically affect students’ higher-order thinking in urban science classrooms are unclear, especially in the absence of empirical research based on multimodal assessment methodologies. This study explored the pathways of these factors by constructing a structural equation model and implemented a functional near-infrared spectroscopy (fNIRS) experiment in an urban science classroom to collect brain data, exploring the mechanisms of students’ higher-order thinking development. The results indicated that both teacher-student relationships and scaffolding instruction could positively influence students’ higher-order thinking, and teacher-student relationships could promote students’ higher-order thinking through the chained mediating role of teacher guidance and scaffolding instruction, while teacher guidance alone had a significant negative effect on students’ higher-order thinking. Furthermore, students’ higher-order thinking during the science class using the scaffolding strategy was related to the increased interpersonal neural synchronization (INS) in the teacher and students’ dorsolateral prefrontal cortex (DLPFC)—a neural basis associated with effective teacher-student interaction. These findings confirm the importance of the teachers’ role during students’ higher-order thinking development and provide evidence from both the structural equation modeling and brain science for the mechanisms behind higher-order thinking development.