The metaverse enables learners to engage in inquiry and interact within virtual spaces,offering high levels of immersion, interactivity, and presence. However, previous studieshave primarily focused on affective outcomes, and research on learners’ higher-ordercognition and knowledge generation remains insufficient. This study examined the effectsof metaverse-based ecosystem learning on high school students’ biological knowledgegeneration using functional near-infrared spectroscopy (fNIRS) to measure prefrontal cortexactivation. Twenty-seven 11th-grade students at a local high school were participated,with one class assigned to the experimental group (metaverse-based learning, n = 15) andanother to the control group (video-based learning, n = 12). During the pretest and posttest,students completed biological knowledge generation tasks while brain activation wasrecorded. The experimental group showed significantly more activation than the controlgroup in the right dorsolateral prefrontal cortex (DLPFC), frontopolar prefrontal cortex(FP), and bilateral orbitofrontal cortex (OFC), indicating that learners effectively engaged inobservation, question, hypothesis, experimental design, and hypothesis evaluation—keyprocesses of knowledge generation—along with heightened cognitive and emotionalengagement. These findings support the effectiveness of immersive inquiry-based learningand neuroscientifically informed instructional design and highlight the need to examinegeneralizability across diverse learning contexts.