For tissue engineering and regenerative medicine, creating thick and heterogeneous scaffold-based tissue constructs requires deep and precise multicellular deposition. Traditional cell seeding strategies lack the ability to create multicellular tissue constructs with high cell penetration and distribution, while emerging strategies aim to simultaneously combine cell-laden tissue segments with scaffold fabrication. Here we describe a technique that allows for three-dimensional (3D) intrascaffold cell assembly in which scaffolds are prefabricated and pretreated, followed by accurate cell distribution within the scaffold using an image-guided technique. This two-step process yields less limitation in scaffold material choice as well as additional treatments, provides accurate cell distribution, and has less potential to harm cells. The image processing technique captures a 2D geometric image of the scaffold, followed by a series of processes, mainly including grayscale transformation, threshold segmentation, and boundary extraction, to ultimately locate scaffold macropore centroids. Coupled with camera calibration data, accurate 3D cell assembly pathway plans can be made. Intrascaffold assembly parameter optimization and complex intrascaffold gradient, multidirectional, and vascular structure assembly were studied. Demonstration was also made with path planning and cell assembly experiments using NIH3T3-cell-laden hydrogels and collagen-coated poly(lactic-co-glycolic acid) (PLGA) scaffolds. Experiments with CellTracker fluorescent monitoring, live/dead staining, and phalloidin-F-actin/DAPI immunostaining and comparison with two control groups (bioink manual injection and cell suspension static surface pipetting) showed accurate cell distribution and positioning and high cell viability (>93%). The PrestoBlue assay showed obvious cell proliferation over seven culture days in vitro. This technique provides an accurate method to aid simple and complex cell colonization with variant depth within 3D-scaffold-based constructs using multiple cells. The modular method can be used with any existing printing platform and shows potential in facilitating direct spatial organization and hierarchal 3D assembly of multiple cells and/or drugs within scaffolds for further tissue engineering studies and clinical applications.