Objective: This study aims to evaluate the application value of biological monitoring and different types of chemical indicator cards in batch monitoring of hydrogen peroxide low-temperature plasma sterilization. The goal is to standardize the selection of loading conditions for this sterilization method and avoid positive biological monitoring results. Methods: Physical monitoring, Class I chemical indicator card monitoring, Class IV chemical indicator card monitoring, and biological monitoring were used to monitor the hydrogen peroxide low-temperature plasma sterilization process. The sterilization effect on instruments inside the Johnson & Johnson 100S plasma sterilizer was monitored and the qualification of various monitoring methods was compared. Results: The comparison showed that when non-standard or adsorption-prone packaging materials were used, the interception rate of biological monitoring and Class IV chemical indicator cards was significantly higher than that of physical monitoring and Class I chemical indicator cards. These methods more intuitively and effectively detected sterilization failures. Conclusion: Biological monitoring and Class IV chemical indicator cards are safe, fast, accurate, and easy to interpret in hydrogen peroxide low-temperature plasma sterilization, especially for monitoring instruments inside packages. They provide a reliable basis for the release of sterilized instrument packages. Identifying the reasons for positive biological monitoring results in hydrogen peroxide low-temperature plasma sterilization and taking effective measures promptly can minimize associated risks.