The integration of bioreceptors with biocompatible substrates is crucial for advancing in vitro microphysiological systems used in disease modeling, drug screening, and biological research. Expanding spatial control over 3‐D bioreceptor patterning enables localized analyte detection, targeted molecular release, and selective sequestration. This study presents strategies for high‐resolution, multiplexed aptamer patterning within hydrogels, achieving the smallest 3‐D aptamer features reported to date (≈2 µm). Aptamers, synthetically engineered single‐stranded DNA or RNA, offer small size, high target specificity, and ease of chemical modification for covalent hydrogel integration. As a proof of concept, two DNA‐based aptamers targeting serotonin and dopamine were immobilized in a norbornene‐functionalized polyvinyl alcohol hydrogel. Systematic evaluation of UV photopatterning, digital light processing, and two‐photon polymerization enabled multiplexed, 3‐D aptamer patterns with micron‐scale resolution. This work establishes a framework for spatially resolved aptamer localization within 3‐D hydrogels, which is particularly important for biosensing in complex in vitro environments, where referencing specific binding requires precise positioning of control DNA near specific aptamers. These advances in spatially controlled aptamer functionalization open new possibilities for engineering modular biointerfaces.