3D printing technology combined with thermoplastic polyurethane (TPU) is increasingly being used in flexible electronics. However, the defects induced by 3D printing may degrade the mechanical properties of the TPU substrates significantly and result in the decrease in breaking elongation, tensile strength, and waterproof performance. In this article, we investigate the effect of heat treatment on improving the mechanical and waterproof properties of TPU substrate experimentally. The results show that heat treatment can effectively reduce the defects such as bubbles and pores inside the 3D printed TPU structure and increase the bonding area of the filament, and thus making the 3D printed TPU structure dense. After heat treatment at 180 C, the tensile strength and elongation at break of 3D printed TPU samples in the fiber direction (filling angle of 0 ) increased by 67% and 38%, respectively. The tensile strength and elongation at break in the bonding direction (filling angle of 90 ) increased by 100% and 75%. In addition, the waterproof performance of the samples has been enhanced, and the water contact angle of the surface has increased from 90.4 to 95.25 . Therefore, the heat treatment can effectively improve the mechanical properties and waterproof performance of 3D printed TPU samples.