Flexible and transparent surface-enhanced Raman scattering (SERS) substrates have attracted considerable attention for their ability to enable the direct in situ detection of analytes on curved surfaces. However, the curvature of an object can impact the signal enhancement of SERS during the measurement process. Herein, we propose a simple approach for fabricating a curvature-insensitive transparent SERS substrate by depositing silver nanoparticles (Ag NPs) onto a large-area wrinkled polystyrene/polydimethylsiloxane (Ag NP@W-PS/PDMS) bilayer film. Using rhodamine 6G (R6G) as a probe molecule, the optimized Ag NP@W-PS/PDMS film demonstrates a high analytical enhancement factor (AEF) of 4.83 × 105, excellent uniformity (RSD = 7.85%) and reproducibility (RSD = 3.09%), as well as superior mechanical flexibility. Additionally, in situ measurements of malachite green (MG) on objects with diverse curvatures, including fish, apple, and blueberry, are conducted using a portable Raman system, revealing a consistent SERS enhancement. Furthermore, a robust linear relationship (R2 ≥ 0.990) between Raman intensity and the logarithmic concentration of MG detected from these objects is achieved. These results demonstrate the tremendous potential of the developed curvature-insensitive SERS substrate as a point-of-care testing (POCT) platform for identifying analytes on irregular objects.