In this work, we have mainly studied SERS spectra of fresh human urine by using Au nanoparticles excited by 785 and 1 030 nm lasers, respectively. And the UV/Vis adsorption experiment of the Au nanoparticles mixed with different ratio of urine has been performed, and the obvious shifting of corresponding absorption band is observed. The result showed that the Au nanoparticles which have been synthesized by classical Fren’s method can interact with urine, and the Au nanoparticles aggregations caused by the urine have strong SERS effect. Intense and repeatable spectra of the urine samples can be quickly obtained using Au colloids, which characterized by the scanning electron microscope (SEM) and the high-resolution transmission electron microscope (HRTEM) images, and it can be confirmed that the size of the Au nanoparticles is about 55 nm with a finite variation. When different spectra can be detected under different exciting lasers, the various biofluid to Au substrate ratios can generate different intense spectra. From the spectra of 785 nm laser, we can conclude that it has lower background and higher resolution with more detail information of this system contained human urine. For the 1 030 nm laser, a portable Raman instrument is helpful for on-site clinic treatment detection. It also gets well defined information and will be a good and convenient choice for urine analysis. It should note that this peak band located at 1 006 cm-1 may be the dominant nitrogen-containing component in urine. On the other hand, uric acid, urea, hypoxanthine as well as creatinine can be assigned; the other bands are still unknown, which might be attributed to biomarkers important for disease differentiation. Another result shows that different sample preparation can influence the SERS spectra with different ratio. We also have made a comparison of Raman spectra between 785 and 1 030 nm lasers to learn the difference between each other just like background and high-resolution. The current study indicates the SERS of urine might be a good choice and tool for urinalysis with potential diagnostic application, especially with the portable Raman instrument which would be an accurate and convenient approach for urine analysis. It is possible for SERS detection to be applied in not only the health diagnosis but also biological tissue in the future.