Surface enhanced Raman scattering (SERS) is a technique that utilizes the surface electric field of metal nanostructures to amplify Raman signals and promote the analysis and detection of trace molecules. In order to harness the potential of flexible SERS substrates, this work proposes a simple and cost-effective method for preparing a large-area (circular, 7.5 cm in diameter, with a 38 cm 2 area) three-dimensional (3D) flexible SERS substrate. We first prepared a bowl-shaped nanoarray of polydimethylsiloxane (PDMS) by utilizing a polystyrene microsphere (PS) as sacrificial templates and then loaded a monolayer of gold nanoparticles (AuNPs) on it, finally a high-strength 3D AuNPs/PDMS flexible SERS substrate is obtained successfully. There are two key steps here, which are using plasma etching to regulate the gap between PS spheres and using the thermal demolding method to facilitate the separation of PS and PDMS. The ultralarge area AuNPs/PDMS substrate exhibits high SERS detection sensitivity and good uniformity. The SERS substrate possesses the advantage of multifunctional detection. The proposed substrate demonstrated detections of up to 10 −9 , 10 −7 , and 10 −8 M concentrations of rhodamine 6G (R6G), vitamin C, and thiram molecules, respectively. The detection results meet international standards. Electromagnetic simulations using the finite difference time domain reveal that the SERS enhancement originates from the surface plasmon resonance and coupling effects of AuNPs. This large-area 3D flexible SERS substrate not only enhances detection efficiency by enabling the detection of a greater number of samples or sample regions but also provides a new approach for detecting vitamin C and pesticide residues. It may also be used in electronic skin sensors, in situ pesticide residue detection, on-scene evidence collection in criminal investigations, and so on.