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In the field of surface‐enhanced Raman scattering (SERS), advances in nanotechnology and surface chemistry have contributed to fabricating the metal substrates with highly sophisticated architectures and strong binding affinity to target molecules which enhanced the sensitivity to target molecules. However, the elaborate yet complicated steps for the synthesis, patterning, and surface modification of metal substrates have often resulted in compromising the reliability, reproducibility, and reusability as SERS substrates. Here, a fully programmable and automated digital maskless flow microlithography process that spatiotemporally controls the fluid flow, UV irradiation, and the shape and location of SERS polymer matrix is provided to fabricate a reliable, reproducible, and reusable hydrogel‐based 3D SERS substrate. The SERS substrates are located inside the microfluidic device in the form of disk‐shaped hydrogels. By rationally designing the functional group chemistry of the hydrogel microposts, Ag nanoparticles are homogeneously synthesized in situ, a target molecule is amplified by 25‐fold inside the microposts, and an enhancement factor as high as 2.4 × 108 is observed. Furthermore, a highly reusable multitarget sensing capability is demonstrated by a sequential analysis of multiple analytes without the trace of former analytes via the intermittent washing step. [ABSTRACT FROM AUTHOR]