Nondestructive investigation and analysis of the internal structure of three-dimensional (3D) bio-printed hydrogel scaffolds is very important for the design, fabrication, and biological functions of engineered tissue scaffolds. Swept- source optical coherence tomography (SS- OCT) is applied to nondestructive imaging and quantitative analysis of 3D bio- printed hydrogel scaffolds. 3D bio- printing technique is used to produce four representative designed geometries of hydrogel scaffolds. High-resolution SS-OCT visualizes the three-dimensional internal structure of the scaffolds, identifies the difference between the designed model and as- produced construction, and detects the printed defects such as fused pores, deformation of pores, breakages of flow channels. The automatic imaging analysis algorithms are developed to quantitatively characterize the pore size (PS) and its distribution, strut size (StS), volume porosity (VP), and pore interconnectivity (PC). It concludes that OCT may be a key tool for the design optimization, process refinement, function realization of 3D bio- printed hydrogel scaffolds.