The purpose of this study was to investigate the area of interest of goalkeepers when defending against kicks from the penalty mark. A remote eye-tracking device and two force plates were used to capture the fixation track and timing of soccer ball diving motions when goalkeepers defend. Ten participants were selected for comparison from grade-1 university soccer league (elite group) and grade-3 university soccer league (ordinary group). The participants watched shooting video in the laboratory, decided the direction of ball and made actual diving action. Two participants from the same group took turns to dive for 10 times of ball shooting in accordance with the official rule of kicking from the penalty mark. Chi-square test for homogeneity and independent sample t test were used. Findings indicated that: 1. The success rate of the elite group for ball diving was significantly higher than the ordinary group. 2. In successful ball diving, the proportion of visual concern over area of interest was also significantly higher than that of wrong direction prediction in elite group. 3. Elite group players mainly gazed at the non-kicking leg and the leg area to kick the ball (23.8%, 4.6%) during running up. On average, before kick off the ball, elite players used 416 ms in deciding which side to dive, which was shorter than the ordinary players, who used 576 ms. However, the differences were not statistically significant different. These data indicates that during the running up, maintenance of concentration is the factor affecting successful soccer ball diving. Pre-cues can be searched based on the observation on players' kicking and non-kicking leg; or on the area before kicking the ball in advance. Nevertheless, it is also necessary to possess rapid diving technique to strive the time for searching pre-cues. During defense practice, it is suggested that multimedia system can be included to increase the reality simulation to train and improve experience of prediction the ball direction.