Porous collagen–based scaffolds are biostructures with demonstrated ability to induce regeneration in animal models as well as in human patients. In addition to their porous structure and physicochemical properties that have been shown to affect the regeneration process, channel-like features inside the scaffolds could provide additional beneficial effects to cells since they provide favorable orientation guidance to cells and favor the transport of required nutrients. Among other methods, ablation by ultra-short laser pulses can be used to fabricate smooth high-precision micro-scale patterns in biomaterials due to their confined damage region. This work presents a holistic approach for the precise generation of complex micron-sized features in porous collagen-based scaffolds, implemented via computer-driven 2½D layer micromachining by a focused femtosecond laser. Tool paths in G code format are generated by a computer program which enables the fabrication of geometric features (straight lines, circular and curved shapes) at specific planes. By taking into account the dependence of ablation volume on key process parameters, such as mean power, feed rate, and focal depth, complex features were fabricated on top and beneath the surface of porous scaffolds at ± 30 μm maximum dimensional tolerance of using optimized cutting parameters.