The aim of this study is to optimize the robustness and controllability of the 3D printed hydrogel scaffolds by iteratively reducing the mismatch between the designed and the as-printed. A feedback loop approach based on optical coherence tomography (OCT) in vivo online quantitative evaluation was performed for twice. The experimental results show that OCT has quantitatively characterized the morphological parameters, and the difference correlation analysis based on the characterization of OCT feedback controls the 3D printing process and enables decrease of the mismatch. The mismatch of the averaged pore size of the hydrogel scaffolds has decreased from 30% to 2%. It concludes that OCT can further expand its applications in the field of tissue engineering, and may be a key tool for scaffold design and characterization, 3D bio-printing process control, and so on.