Located on the right side of the heart, the tricuspid valve (TV) prevents blood backflow from the right ventricle to the right atrium. Similar to other cardiac valves, quantification of TV biaxial mechanical properties is essential in developing accurate computational models. In the current study, for the first time, the biaxial stress–strain behavior of porcine TV was measured ex vivo under different loading protocols using biaxial tensile testing equipment. The results showed a highly nonlinear response including a compliant region followed by a rapid transition to a stiff region for all of the TV leaflets both in the circumferential and in the radial directions. Based on the data analysis, all three leaflets were found to be anisotropic, and they were stiffer in the circumferential direction in comparison to the radial direction. It was also concluded that the posterior leaflet was the most anisotropic leaflet.