Abstract: In order to reduce diseases (such as lung cancer) incidence and mortality, a key work is early diagnosis and treatment, in which an acupuncture treatment biopsy is a common means of treatment. To solve the low accuracy and high probability of repeat surgery caused by manpower acupuncture, we need to design a high precision surgical navigation system. Before the system can be used, the relative positions of the system component should be calibrated. To calibrate the relative positions between needle, robotic arm and binocular stereo vision for subsequent surgical navigation, this paper proposes a system calibration method based on visible markers. According to the experimental result, the needle tip calibration error is 1.37mm with an attitude error of 0.62°, which meet the accuracy of thoracic surgery.