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In this article, a high-performance nanostructured substrate has been fabricated for the ultrasensitive detection of the organic pollutant, Malachite green isothiocyanate (MGITC), in aquatic systems via the Surface Enhanced Raman Spectroscopy (SERS) technique. The chemical dealloying approach has been used to synthesize a three-dimensional nanoporous gold substrate (NPG) consisting of pores and multigrained ligament structures along thickness. The formation of the framework in NPG-5h has been confirmed by SEM with an average ligament size of 65 nm at the narrower neck. Remarkable SERS performance has been achieved by utilizing the NPG-5h substrate for the detection of MGITC, showing a signal enhancement of 7.9 × 109. The SERS substrate also demonstrated an impressively low-detection limit of 10−16 M. The presence of numerous active sites, as well as plasmonic hotspots on the nanoporous surface, can be accredited to the signal amplification via the Localized Surface Plasmon Resonance (LSPR) phenomenon. As a result, SERS detection technology with the fabricated-NPG substrate not only proves to be a simple and effective approach for detecting malachite green but also provides a basis for in situ detection approach of toxic chemicals in aquatic ecosystems.