The accurate positioning of the core DNA nanostructure is one of the crucial problems in constructing nano-devices or nano-systems with the controlled self-assembly of the specified location DNA nanostructure.A programmable control AFM manipulation method was studied. Through the parameter control in the process of the manipulation, the controllable positioning and orientation manipulation of the flexible DNA nanostructures in the liquid environment were achieved. The experiment optimization for the two key parameters of the manipulation amplitude and Mg2+ concentration in the liquid was carried out. The DNA nanotube structure with the length of 400 nm and the diameter of about 6 nm was adopted in the experiment. The experiment results show that the positioning and orientation manipulation of the DNA nanostructure in the liquid environment is realized with the programmable controlled AFM manipulation method under the optimized conditions of 3.5 nm manipulation amplitude and 10 mmol/L Mg2+concentration, and the success rate reaches 80% .