With cutting-edge technologies and considering airline human-resource-saving, a single pilot in commercial jets could be technically feasible. Investigating changes in captains’ natural behaviours are initially required to comprehend the specific safe human performance envelope for safeguarding single-pilot flight, particularly in high-risk situations. This paper investigates how captains’ performance transforms for fixing emergencies when operating from Dual-Pilot Operations (DPO) to Single-Pilot Operations (SPO) through a physiological-based approach. Twenty pilots flew an emergency-included flight with/without first officers’ assistance. The neural activities and scanning behaviours were recorded using a 32-channel Electroencephalogram (EEG) and glasses-based eye tracker, with the observation and post-experiment questionnaires to evaluate the flight operations and pilots’ perception. Flying alone, there was a significantly increased cortical activity in θ and β waves over the frontal, parietal, and temporal lobes during the more complicated emergencies, and pilots focused less on the primary flight display while spending significantly more time scanning the other interfaces. The physiological fluctuating patterns associated with risky operations in SPO were highlighted by cross-correlating multimodal data. The experimental-based noteworthy insights may wish to inform commercial SPO measures to lessen the persistent physiological fluctuation, assisting airlines in creating SPO-oriented intelligent flight systems to give captains adequate support for assuring safer air transportation.