The invention discloses a big gecko's vertebral column three-dimensional positioning device and a big gecko's vertebral column three-dimensional positioning method applied to big gecko in-vivo spinal cord preparation and electrophysiological related experiments thereof, and belongs to the technical field of positioning. According to the method, by using the characteristic that a concave arc structure between the front and rear articular processes of the cervical vertebra, the thoracic vertebra and the lumbar vertebra of a big gecko can be used for clamping and positioning, a clamping device is designed to clamp two adjacent vertebras, so that different zones of the vertebral column of the big gecko are reliably positioned. The device consists of a supporting seat, a moving block, a vertebra clamp, a set screw and a locking screw. The device is reliable in positioning and easy to operate, fills the blank that a special vertebral column three-dimensional positioning device for gecko reptiles does not exsit at home and abroad, and lays a foundation for the big gecko in-vivo spinal cord preparation and electrophysiological experimental study thereof.