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Nowadays, chronic kidney disease (CKD) becomes a principal barrier in clinical diagnosis and treatment. For clinical patients, chronic kidney disease will potentially lead to multiple lesion that increase the risk of mortality. The most significant challenge in CKD is detecting uremic toxins, including small water-soluble solutes (uric acid, urea and creatinine) and protein-bound solutes (p-cresol and indoxyl sulfate). Surface-enhanced Raman scattering (SERS) platform is rapid and sensitive nanotechnology for bio-detection. Thus, the floating-typed SERS substrate is prepared by embedded silver nanoparticles (AgNPs) on the poly (diallyldimethyl-ammonium) chloride (PDDA) modified graphene oxide (GO) nanosheets for the biomolecules and uremic toxins detection. The optimal interparticle distances of AgNPs are modulated to generate the strong “hot spots” for enhancing Raman signals. The characterizations of AgNPs/GO-PDDA nanosheets are evaluated by transmission electron microscopy, zeta potential, FTIR, X-ray photoelectron spectroscopy, and Raman spectroscopy. The results show that the floating SERS-active substrate provides quantitatively linear measurement (i.e., 3.8 × 10−2 ˜10-5 M of urea) and ultrasensitive SERS detection (i.e., detection limit of adenine: lower than 10-10 M) of biomolecules, which offers great potential for practical clinical applications in rapid and label-free detection of clinical uremic toxins.