A lack of situational awareness has been attributed as one of the major factors in shipping accidents. The importance of situational awareness has also been consistently highlighted in studies investigating the required skills of autonomous ship operators. Consequently, a growing body of literature has been dedicated to studying seafarers’ situational awareness. Most of the studies, if not all, use ships or ship simulators to investigate seafarers’ situational awareness. This study proposes a simple and cost-effective method to evaluate seafarers’ situational awareness by using bio-signals and a relevant ship operational video. Specifically, this study employs heart rate variability and ocular behavior and a video of a traffic route operation. A novice seafarer and an experienced seafarer are shown with a video of a southbound vessel passing through the Uraga Channel. As a result, both respondents show similar trends of heart rate variability and ocular behavior, which imply a similar level of situational awareness despite having substantial differences in terms of seafaring experience. The reason behind this is mainly due to the straightforwardness of the traffic route operation. In the future, bio-signal data from respondents performing a similar navigational task using ships or ship simulators are required to further support the findings of this study. As important, more respondents are also needed to confirm the usability of the proposed method.