Low-frequency ultrasound has been extensively utilized in transdermal drug delivery, effectively improving drug penetration. The effects of 20 kHz low-frequency ultrasound with varying duty cycles (25%, 50%, 75%, and 100%) on the temperature of biological tissues were comprehensively analyzed to explore the safety of ultrasound transdermal drug delivery in this paper. Based on the findings from finite element analysis and physical modeling using COMSOL Multiphysics software, there exists a direct correlation between the duty cycle of the ultrasound transducer and the temperature of biological tissues. Specifically, the temperature of the biological tissues also increased with an increase in the duty cycle. When the ultrasound duty cycles above 50%, the highest temperature within the biological tissue exceeds 42 °C, failing to meet the safety standards for ultrasound transdermal drug delivery. These findings can provide valuable guidance for configuring duty cycle parameters in low-frequency ultrasound transdermal drug delivery procedures.