Display omitted •Aromatic amphiphilic block copolymer named of amPEN has been synthesized.•AmPEN based micro-reactors are obtained via reverse emulsion self-assembly.•Silver nanoparticles (Ag NPs) are synthesized in the obtained micro-reactors.•Ag NPs are self-assembled into ~ 300 nm cube-like plasmonic superstructures (PSP).•Chlorpyrifos is detected via the optimized PSP SERS substrate. Hierarchical anisotropic superparticles derived from the controlled assembly of individual plasmonic nanoparticles have been recognized as promising building blocks for surface-enhanced Raman spectroscopy (SERS) nanosensors. Thus, the protocols that precisely control the near-field coupling of individual plasmonic nanoparticles and the diffusion of highly diluted analytes to the plasmonic surface are of great interests. Herein, we have synthesized a series of aromatic amphiphilic block copolymers named as polyarylene ether nitrile (amPEN in short), containing a hydrophobic backbone of aromatic moieties with hydrophilic sulfonate/carboxyl and cyano groups in the side chain. Next, the surfactant stabilized water-in-oil (W/O) reverse emulsion is constructed to induce the confined self-assembly of amPEN, leading to the formation of functional micro-reactors for the in-situ synthesis and organization of silver nanoparticles, which finally contribute to the generation of sub-micrometer cube-like plasmonic superparticles (PSPs). Furthermore, the SERS nanosensors are created by drop-casting of synthesized PSPs on silicon substrate followed by depositing an additional thin layer of silver. Finally, the optimized SERS nanosensors with a Raman enhancement factor of 108 are able to detect chlorpyrifos pesticide down to 10-10 M thanks to the presence of abundant Raman hot-spots on the synthesized PSPs and the effective analyte enrichment derived from π-π interaction between pesticide molecules and aromatic backbone of amPEN.