We report on laser-assisted fabrication and non-invasive imaging of porous 3D cell-seeding constructs (3D-CSCs) for bone tissue engineering. The 3D structures were built by two-photon polymerization-direct writing (2PP_DW) of IP-L780 photopolymer and consist in arrays of vertical microtubes arranged in triangular lattices. The microtubes were tightly, medium, and rarely packed, according to the constants of the triangular lattices of 8, 12, and 24 mu m, respectively. The efficiency of the laser-generated 3D-CSCs for new bone formation was assessed in MG63 osteoblast-like cells cultures. High spatial resolution 3D images of the cell-seeded 3D-CSCs were obtained by digital holographic microscopy (DHM). The recorded holograms allowed the simultaneous evaluation of the 3D-CSCs and of the seeded cells, in terms of 3D shapes and dimensions, without intruding into the cells natural environment. The seeded cells, in particular the cells nuclei, conformed to the micro-architectures of the 3D-CSCs. Furthermore, the osteogenic potential of the 3D-CSCs was assessed in terms of cell morphology, viability, and level of mineralization. The microtubes packing density that allowed the seeded osteoblasts to reach the highest level of mineralization was established.