Objective To design and prepare a glass-shaped transthoracic left atrial appendage (LAA) reverse occluder and delivery system, and to evaluate the feasibility of transthoracic left atrial appendage reverse occlusion by in vitro reverse occlusion experiment. Methods We used fresh canine heart specimens and measured the anatomical parameters, including the long diameter, short diameter and perimeter of atrial appendage opening, the thickness of upper 1/3 left atrial appendage, the shortest distances from the edge of the opening of the atrial appendage to the left superior pulmonary vein, the left inferior pulmonary vein, and the mitral valve. A new glass-shaped left atrial appendage occluder was designed using nitinol wire, and the appendage occluder opening disk diameter and waist height were designed according to the average diameter of the opening of the atrial appendage and the thickness of upper 1/3 left atrial appendage. The delivery system comprised 9F delivery sheath, expansion sheath, preload sheath and a pushing cable. Delivery sheath was scaled which can be used to control the depth into the left atrial appendage, and expansion sheath head end had smooth transition shape which exposed only about 0.8 cm when inserted into the delivery sheath to prevent damage internal of tissue of heart. Ten isolated dog hearts were punctured at the center of the upper 1/3 axis of left atrial appendage outside under direct vision, and 9F sheath with scale was used to complete in vitro experiment of occlusion. The location and effect of the occluder were observed. Results Nine of the 10 heart specimens successfully underwent the occluding tests in vitro. It was showed that the occluder was well located, with the disc of the opening of left atrial appendage porting into the opening a little and the opening in an overdistraction state, and there was no influence on blood flow of pulmonary vein or function of the mitral valve. The ideal point of puncture was at the upper 1/3 long axis of left atrial appendage outside, and the purse-string suture should focus on this point. The puncture points located at the center of the left atrial appendage opening after removing the occluder postoperatively. Only 1 case failed because of low puncture point and unsuitable location of the occluder. Conclusion The glass-shaped transthoracic reverse occluder is well-designed and matched the anatomy of left the atrial appendage in experimental canine. The occluding effects are exact and meet the requirements of animal experiments in vivo.