Creatinine level in urine is an important biomarker for renal function diseases, such as renal failure, glomerulonephritis, and chronic nephritis. The Au@MIL-101(Fe) was prepared by in situ growth of Au nanoparticles in MIL-101(Fe) as a selective SERS substrate. The Au@MIL-101(Fe) offers the great local surface plasmon resonance (SPR) effect due to gold nanoparticles aggregation inside metal-organic frameworks. The framework structure could enrich trace target samples and drag them into SPR hot spots. The optimal Au@MIL-101(Fe) composite substrate is used for analyzing creatinine in urine and the limit of detection is down to 0.1 μmol/L and a linear relationship is ranging from 1 μmol/L to 100 μmol/L.