Development of high-performance flexible substrates for surface-enhanced Raman spectroscopy (SERS) is crucial for the sensitive, rapid detection of environmental pollutants in complex matrices. In this work, we report the fabrication of hydrothermally synthesized nickel oxide microflowers decorated with photoreduced silver nanoparticles on filter paper (Ag-NPs/NiO-MFs@FP) for the detection of the priority pollutant 4-nitrophenol (4-NP). The unique 3D hierarchical structure of the NiO MFs provides a wide surface area for the deposition of high-density Ag-NP and 4-NP molecules. The formation of numerous hotspots in the nanoscale gaps between adjacent Ag NPs produces a significant Raman signal enhancement. Furthermore, the energy band structure of Ag NPs and NiO MFs facilitated the efficient electron transfer to the 4-NP molecules, which further amplifies the SERS effect. The Ag-NPs/NiO-MFs@FP SERS substrate exhibits excellent performance for the detection of 4-NP, including an ultra-low limit of detection (LOD) of 3.38 × 10−12 M, an enhancement factor of 3.31 × 109, good reproducibility (RSD of 7.62 %), high uniformity, and remarkable long-term stability (70 % efficiency after 12 days). Importantly, the flexible SERS platform enables the sensitive and reliable detection of 4-NP in tap water and river water, and on fish surfaces with good recovery rates. The performance comparison results show that the proposed Ag-NPs/NiO-MFs@FP composite has superior detection performance compared to other SERS active materials and other detection methods for the detection of 4-NP. The facile fabrication approach, superior analytical capabilities, and practical feasibility of the Ag-NPs/NiO-MFs@FP SERS substrate make it a highly promising detection platform for efficient environmental monitoring and food safety applications.