Three-dimensional imaging of thick tissue constructs is one of the main challenges in the field of tissue engineering and regenerative medicine. Optical methods are the most promising as they offer noninvasive, fast, and inexpensive solutions. Herein, we report the use of mesoscopic fluorescence molecular tomography (MFMT) to image function and structure of thick bioprinted tissue hosted in a 3-mm-thick bioreactor. Collagen-based tissue assembled in this study contains two vascular channels formed by green fluorescent protein- and mCherry-expressing cells. Transfected live cell imaging enables us to image function, whereas Flash Red fluorescent bead perfusion into the vascular channel allows us to image structure. The MFMT optical reconstructions are benchmarked with classical microscopy techniques. MFMT and wide-field fluorescence microscopy data match within 92% in area and 84% in location, validating the accuracy of MFMT reconstructions. Our results demonstrate that MFMT is a well-suited imaging modality for fast, longitudinal, functional imaging of thick, and turbid tissue engineering constructs.