Compared to other fabrication methods such as micromolding [7] or soft lithography, [8] objects manufactured via FDM are produced in their final shape without any external template, additional tools, or further postprocessing. Since FDM does not require any mastermold, it allows the fabrication of complex, tailorable lattices with high reproducibility, such as highly hollow or gyroidal structures. [9] FDM feedstock is commodity plastics, avoiding the use of hazard or expensive compounds. Moreover, FDM printers are more economic and relatively easy to use than most of other (micro)fabrication equipment. The printed objects are previously designed using a software and all the information needed for fabrication is contained within a computer assisted design file. These designs can be easily modified to meet the quick changing needs of the market, allowing high flexibility in the product and reducing the product development process. [10] Since 3D printing technologies emerged, polylactic acid (PLA) has become one of the most widely used materials in the FDM technology due to its cost, biodegradability, and good mechanical properties. [11] PLA can be manufactured from natural sources as sugarcane or corn starch and possesses mechanical properties comparable to fossil-fuel derived polystyrene. [12] Among its limitations, it presents a low maximum service temperature (≈60 °C), which narrows its use in engineering applications. [13] However, it can be used as structural matrix when working at room or human body temperature. Bio-based polymers, including PLA, have been printed to fabricate tablets, [14] scaffolds, [15] or even organs individually adapted for each patient. [16] In addition, since additive manufacturing techniques emerged, there is a growing number of initiatives to fabricate different equipment and laboratory supplies in house, [17] which would allow reducing the production chain, saving time and money. Another important factor to consider in the design of micro total analysis systems is the surface modification of the material so it can selectively adsorb the target biomolecule. Among other alternatives, the LbL technology is an interesting option which allows the modification of large surfaces in an inexpensive manner. [18-20] This technology is known since 1997 [21] and consists in the fabrication of thin films via electrostatic or supramolecular interactions of different polymers, which allows In this paper a methodology is presented to fabricate a micropatterned device made of bio-based polymers (polylactic acid, sodium alginate (ALG), and chitosan (CHI)) suitable for micro total analysis. Fused deposition modeling and layer-by-layer technologies are combined to create tailorable devices with submillimetric controlled well size that can be rapidly prepared in house. As proof of concept, the influence of immobilization of ALG/CHI bilayers in the selective adsorption of alkaline phosphatase and its activity is studied. Due to the noncovalent nature of these interactions, it is proven that these devices ...