•Ag nanoparticles dispersed on UF microsphere is used as an effective SERS substrate.•Excess formaldehyde in UF synthesis is crucial for preparation of the substrate.•Drying and incubation methods of the substrate affect the SERS performance.•Various inorganic salts modify the substrate basing on their affinity with silver.•Ag nanoparticles dispersed on UF microsphere has a R6G detection limit of 10−13 M.Urea and formaldehyde (UF) microsphere (MS) adsorbing Ag nanoparticles (NPs) was employed as a surface enhanced Raman scattering (SERS) substrate for rhodamine 6G (R6G) detection. The UF MSs and citrate-reduced Ag colloid supplying Ag NPs are synthesized separately and all the subsequent fabrication procedure is then implemented within 2 mL centrifuge tube. Influences of the composition and drying temperature of the UF MSs and the drying method and modification of AgNP/UFMS on the final SERS performance have first been reported. Excess formaldehyde useful in the formation of UF MSs again plays an important role in the SERS detection. Some interesting phenomena in the approach, such as swelling/deswelling of UF MSs and R6G diffusion within hydrophilic environment of UF MSs, are found to be of variable factors affecting the SERS performance. The substrate AgNP/UFMS confidently achieves a detection limit of 10−13 M R6G and can be used as a simple and effective platform in the SERS spectroscopy.