The present invention provides a positioning device and method for puncture of endoscope operated surgery robot. The device comprises an expansible connection rod arranged at end of a mechanical arm, a miniature CCD camera is arranged at end of the expansible connection rod, two red light point-like light spot lasers are arranged under the end mechanical arm of the robot, light from the both red light point-like light spot lasers intersects at front lower part of the camera, the red light point-like light spot lasers and the miniature CCD camera connect with control industry PC machine. The invention improves calibration precision of the puncture, conserves calibration devices such as CCD camera, prevents slow system operation caused by occupation of considerable computer system resource when only executing double-target or multi-target calibration in three-dimensional reconstruction process, thus the puncture calibration process is improved to be rapid and accurate, thereby reducing cost and being convenient to develop and produce products.