Flexible conductive skin patches were readily fabricated on silk fabric by in situ deposition of gold nanoparticles (AuNPs) followed by carbonization. The carbonized AuNPs-silk with high flexibility was characterized by field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and Fourier transform Raman spectroscopy (FT-Raman) to verify the well arrangement surface and desired chemical binding. The conductivity of silk skin patch, measured by a four-point probe, was found to be 109.24 ± 13 S cm−1 × 10−3, verifying the potential application as a working electrode in electrochemical sensor and a sweat collection patch for direct detection by laser desorption/ionization mass spectrometry (LDI-MS). This silk skin patch offered a linear range of 0–100 mM with a detection limit (LOD) of 20 mM for electrochemical sensor and 8 mM for LDI-MS, respectively. Ultimately, this skin patch is successfully applied for the detection of sweat urea at its cut-off value (60 mM) for indicating chronic kidney disease (CKD) in artificial sweat with satisfactory results. By using dual-detection technique on single silk substrate, this platform might be an alternative approach for a non-invasive sweat urea detection with high precision.