The dynamic regulation of neurotransmitter secretion and its in situ detection can provide valuable insights into the core mechanisms underlying neural network formation and function. Current techniques for detecting neurotransmitter secretion suffer from limitations such as low sensitivity, susceptibility to biomolecular contamination, and a lack of capability for in situ monitoring. To address these challenges, we developed a three-dimensional gold nanonetwork-based surface-enhanced Raman scattering (SERS) substrate fabricated through layer-by-layer self-assembly using alternating deposition of PDDA solution and AuNPs. This structure utilizes charge balance and hierarchical stacking of gold nanoparticles to generate high-density electromagnetic “hotspots” via localized surface plasmon resonance (LSPR), achieving an enhancement factor on the order of 10⁶ in the detection of 4-ATP. Remarkably, the as-prepared substrate enables label-free dopamine detection at concentrations as low as 1 nM without requiring surface modification. Furthermore, we constructed magnetically responsive cells using FDA-approved ferumoxytol, which were directly cultured on the SERS substrate. Under magnetic stimulation, the release of cellular nucleic acids, amino acids, and lipids could be monitored. Based on these findings, the SERS-based detection platform we developed holds great potential for establishing a novel methodological framework that integrates magnetic stimulation and SERS spectroscopy to elucidate mechanoregulated neurotransmitter secretion events.