High-quality biosecurity practices are critical to restarting international tourism. Effective market segmentation improves the communication and efficacy of health advice. Travel frequency is an important basis for health-related consumer segmentation, as it is closely related to risk of greater exposure to infectious diseases. Theoretically grounded studies of tourist biosecurity behavior and travel frequency have largely been neglected, although insights into practices and attitudes are especially relevant for coronavirus disease of 2019 (COVID-19 (coronavirus disease of 2019) health responses. Therefore, this research constructed and tested a conceptual model applying Value–Attitude–Behavior theory to US travelers to see whether the frequency of international travel affected tourist COVID-19 related biosecurity behavior. US respondents were drawn from a panel using a quota sampling technique according to the age and gender of American outbound tourists. An online survey was administered in September 2020. The responses (n = 395) of those who traveled internationally within five years were analyzed utilizing partial least squares-structural equation modeling (PLS-SEM) with multi-group analysis. Travel frequency significantly affects biosecurity behavior. High travel frequency (≥8 trips) has the strongest effect of value on biosecurity attitudes, personal norms, social norms, and biosecurity social norms, leading to biosecurity behaviors. Biosecurity behaviors pertaining to medium travel frequency (4–7 trips) are significantly influenced by personal norms. At low travel frequency (1–3 trips) levels, biosecurity behaviors are stimulated by biosecurity attitudes and social norms, showing the highest predictive power among the three groups. This work provides insights into international travel consumer biosecurity practices and behavior. From a market segmentation perspective, the levels of international travel frequency have various influences on biosecurity values, attitudes, personal norms, social norms, and behaviors. The biosecurity behaviors of low-frequency travelers are found to be the most significant of the three groups, suggesting that individuals who travel less frequently are more likely to practice responsible COVID-19 biosecurity behavior.