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Abstract Surface‐enhanced Raman scattering (SERS) sensing in microfluidic devices, namely optofluidic‐SERS, suffers an intrinsic tradeoff between mass transport and hot spot density, both of which are required for ultrasensitive detection. To overcome this compromise, photonic crystal‐enhanced plasmonic mesocapsules are synthesized, utilizing diatom biosilica decorated with in‐situ growth silver nanoparticles (Ag NPs). In the optofluidic‐SERS testing of this study, 100× higher enhancement factors and more than 1,000× better detection limit are achieved compared with traditional colloidal Ag NPs, the improvement of which is attributed to unique properties of the mesocapsules. First, the porous diatom biosilica frustules serve as carrier capsules for high density Ag NPs that form high density plasmonic hot‐spots. Second, the submicron‐pores embedded in the frustule walls not only create a large surface‐to‐volume ratio allowing for effective analyte capture, but also enhance the local optical field through the photonic crystal effect. Last, the mesocapsules provide effective mixing with analytes as they are flowing inside the microfluidic channel. The reported mesocapsules achieve single molecule detection of Rhodamine 6G in microfluidic devices and are further utilized to detect 1 × 10⁻⁹mof benzene and chlorobenzene compounds in tap water with near real‐time response, which successfully overcomes the constraint of traditional optofluidic sensing.