The synthesis of new surface-enhanced Raman scattering (SERS) substrates for the detection of trace poisonous organic pollutants is important for environmental monitoring. Herein, we designed and prepared monodispersed and uniform Ag NPs anchored on silica microspheres (Ag@ASMs) by the orientation of grafted amino groups and applied it as a SERS substrate for the detection of ultralow concentrations of poisonous organic pollutants (Rhodamine 6G (R6G), crystal violet (CV), and melamine) with high sensitivity and reproducibility in aqueous solution. A ratio of 2:1 Ag@ASMs exhibited optimum SERS performance, allowing the detection of 10(-12) M of R6G with an enhancement factor of 6.36 X 10(7) (collect the normal Raman signal based on 10(-4) M R6G) as well as 10(-8) M of CV and melamine. The density functional theory calculation also provided evidence that modified 3-triethoxysilylpropylamine molecules are interspaced, and the optimum Ag dosage further tuned the gap between Ag NPs as well as the size of Ag NPs. This approach can suggest new ideas for the design and synthesis of other improved SERS-active substrates.