The invention provides an ultrasonic-assisted optothermal agent-based tumor cell killing method which comprises the following steps: (1) providing a target sample, and injecting a medical carrier containing an optothermal agent into the target sample, wherein the target sample is tissue or cell suspension; (2) performing ultrasonic treatment on the target sample (treated in the step (1)) by adopting an ultrasonic device, irradiating tumor cells by adopting near-infrared laser until the temperature of the tumor cells is higher than 43 DEG C, and controlling the output power and frequency of the ultrasonic waves so that the temperature of normal cells is not higher than 43 DEG C. According to the combined therapy provided by the invention, high efficiency of photothermal therapy and characteristics such as continuous temperature rise and high penetrating power in ultrasonic thermotherapy are integrated, chemotherapeutic medicines and photothermal agents in a lower dose can be used under the same treatment effect, and toxic and side effects of the medicines on normal tissues of the body are greatly reduced. The invention also provides an ultrasonic-assisted optothermal agent-based tumor cell killing system and application thereof.