Gluconacetobacter xylinus (G. xylinus) metabolism is activated by oxygen, which makes the formation of an air-medium interface critical. Here we report solid matrix-assisted 3D printing (SMAP) of an incubation medium surface and the 3D fabrication of bacterial cellulose (BC) hydrogels by in situ biosynthesis of G. xylinus. A printing matrix of polytetrafluoroethylene (PTFE) microparticles and a hydrogel ink containing an incubation medium, bacteria, and cellulose nanofibers (CNFs) are used in the SMAP process. The hydrogel ink can be printed in the solid matrix with control over the topology and dimensional stability. Furthermore, bioactive bacteria produce BC hydrogels at the surface of the medium due to the permeability of oxygen through the PTFE microparticle layer. The flexibility of the design is verified by fabricating complex 3D structures that were not reported previously. The resulting tubular BC structures suggest future biomedical applications, such as artificial blood vessels and engineered vascular tissue scaffolding. The fabrication of a versatile free-form structure of BC has been challenged due to restricted oxygen supplies at the medium and the dimensional instability of hydrogel printing. SMAP is a solution to the problem of fabricating free-form biopolymer structures, providing both printability and design diversity.