Spatial and temporal control of bioactive signals in three-dimensional (3D) tissue engineering scaffolds is greatly desired. Coupled together, these attributes may mimic and maintain complex signal patterns, such as those observed during axonal regeneration or neovascularization. Seamless polymer constructs may provide a route to achieve spatial control of signal distribution. In this study, a novel microparticle-based scaffold fabrication technique is introduced as a method to create 3D scaffolds with spatial control over model dyes using uniform poly(D,L-lactide-co-glycolide) microspheres. Uniform microspheres were produced using the Precision Particle Fabrication technique. Scaffolds were assembled by flowing microsphere suspensions into a cylindrical glass mold, and then microspheres were physically attached to form a continuous scaffold using ethanol treatment. An ethanol soak of 1 h was found to be optimum for improved mechanical characteristics. Morphological and physical characterization of the scaffolds revealed that microsphere matrices were porous (41.1 ± 2.1%) and well connected, and their compressive stiffness ranged from 142 to 306 kPa. Culturing chondrocytes on the scaffolds revealed the compatibility of these substrates with cell attachment and viability. In addition, bilayered, multilayered, and gradient scaffolds were fabricated, exhibiting excellent spatial control and resolution. Such novel scaffolds can serve as sustained delivery devices of heterogeneous signals in a continuous and seamless manner, and may be particularly useful in future interfacial tissue engineering investigations.