One of the largely important focuses in Air Traffic Control (ATC) operations is radar-based operation in enroute facilities. Many aspects in enroute facilities including airspace and Air Traffic Controller (ATCO) factors need to be further investigated in dealing with the continous air traffic increase. Moreover, ATCOs mental workload and emotion were two important constructs that need to be continually assessed to deal with the increase. This study aims to examine the effects of different enroute factors including number of crossing, traffic flow, and separation on ATCOs' perceived and physiological mental workload and emotion as well as to assess the relation between ATCOs' physiological and perceived mental workload and emotion. The results revealed that the number of crossing significantly affect ATCOs' mental workload. Regarding the mental workload and emotion assesments, ATCOs perceived mental workload was generally higher than their physiological mental workload. However, ATCOs perceived emotion was more positive than their physiological emotion. Further, high mental workload was highly associated with negative emotion in enroute ATC environment. This study offers several implications for human performance enhancement in future ATC operations including investigation of airspace factors, appropriate assessment methods, as well as ATC system design for positive emotion.