Objective To establish a safe and effective method of rhesus monkey biopsy to take liver and kidney samples under B-mode ultrasound guidance. Methods A total of 4 adult monkeys (weight: 8 -12 kg; sex: male; age: 11 - 12 years old) were anaesthetized with 5 - 10 mg/ kg of ketamine hydrochloride for each through intramuscular injection. After successful anesthesia, abdominal shaving and iodophor disinfection, they were monitored from intercostal area of right upper quadrant or lateral waist subcostal abdomen portions to find liver or kidney organ by MyLab 30CV B-mode ultrasonography with 3.5 Hz transducer which was fixed with a guiding frame. Large vessels such as the portal vein and inferior vena cava were carefully avoided. The range of the biopsy gun was set to 15 mm. When the puncture target and the puncture needle were positioned in the guide line, the puncture target was perpendicular to the puncture needle, and then the trigger button of the puncture needle was pressed to obtain the liver or kidney tissue samples respectively. After puncture, the needle was pulled out quickly. The obtained liver and kidney tissues were used to extract RNA. Results About 13 mg of liver or kidney tissue was obtained by each puncture with volume convertion. This method was fast, reliable and safe, and the total RNA had high purity and integrity. There was no postoperative bleeding and infection. Conclusions This is a very important method for obtaining liver and kidney tissue samples of rhesus monkeys with the guidance of ultrasound. With this method , the research cost can be reduced, the life quality and animal welfare of laboratory nonhuman primates can be improved, and the accuracy of experimental result can be ensured.