To face the increasing demand for organ transplantation, currently the development of tissue engineering appears as the best opportunity to effectively regenerate functional tissues and organs. However, these approaches still face the lack of an efficient method to produce an efficient vascularization system. To answer these issues, the formation of an intra-volume channel within a three-dimensional, scaffold free, mature, and cell-covered collagen microfibre is here investigated through laser-induced cavitation. An intra-volume channel was formed upon irradiation with a near-infrared, femtosecond laser beam, focused with a high numerical aperture lens. The laser beam directly crossed the surface of a dense and living-cell bilayer and was focused behind the bilayer to induce channel formation in the hydrogel core while preserving the cell bilayer. Channel formation was assessed through confocal microscopy. Channel generation inside the hydrogel core was enhanced by the formation of voluminous cavitation bubbles with a lifetime longer than 30 s, which also improved intra-volume channel durability. Twenty-four hours after laser processing, cellular viability dropped due to a lack of sufficient hydration for processing longer than 10 min. However, the processing automation could drastically reduce the cellular mortality, this way enabling the formation of hollowed microfibres with a high density of living-cell outer bilayer.Read less <