This article presents a method for the assembly of millimeter-scale Janus fibers using acoustophoresis as an assembly mechanism. An acoustic flow cell mounted to a 3D printer combines acoustophoresis and additive manufacturing in a unique approach that allows for the assembly of textured Janus fibers. A dispersion consisting of polymethylmethacrylate (PMMA) filler particles in a UV curable polymer resin is passed through an acoustically excited capillary tube. To fundamentally understand this process, we develop a suspension balance model that accounts for acoustophoresis and concentration-driven shear-induced diffusion. Once assembled, the particle-polymer dispersion is cured using UV illumination to create a polymer composite fiber with particles immobilized on one side in a Janus-like configuration. The Janus fiber is observed to modify the light transmission profile when rotated on an optical microscope stage. Tensile measurements of the fiber show that the Young's modulus of the Janus fiber (50.5 MPa) is approximately twice that of a fiber fabricated from the polymer alone (24.7 MPa). The process we describe here could serve as a pathway for the fabrication of a variety of functional Janus fibers with possible applications to wearable textiles, soft robotics or surgical sutures.