In this paper, we used eye tracking methodologies to investigate students’ visual perceptions of lectures using 3D real-time virtual studio technology. For measuring learning performance, we also gave the students multiple-choice paper quizzes at the end of the lectures. Three virtual lectures were created with different types of lecture materials (text-centered, image-centered, and lecturer-centered) and 3D virtual sets (classroom, cyberspace, and lecture-theme space). Through analyzing students’ eye movements in viewing still and moving scenes of the virtual lectures, we found that layouts and movements of design elements on lecture screens significantly influenced students’ scanpaths and areas of interest (AOIs). Lecture material types affected learning performance while 3D virtual sets had no effect due to students’ inattention to the virtual background areas. We discuss effective ways to develop virtual lectures and design lecture screens for better presentation of lecture content and higher learning performance.