An evaporative self-assembly strategy for constructing a long-life 3D surface-enhanced Raman scattering (SERS) hot spot matrix is proposed with the assistance of glycerol to improve the spectral sensitivity and reproducibility. Different from the traditional wetting-state or drying-state methods, silver nanoparticles (AgNPs) in the glycerol-stabilized 3D SERS hot spot matrix can be maintained in the translation state for more than 7 days with the maximal uniformity of the interparticle distance. Brownian dynamics simulations reveal that more hot spots emerge in the glycerol-stabilized AgNPs matrix, and the distances between the AgNPs are not fixed but balanced in a small range by the interplay of the van der Waals attraction and the electrostatic repulsion. A 2 orders of magnitude extra SERS enhancement, more stable peak frequencies, and a narrower peak full width at half-maximum (fwhm) can therefore be obtained, which ensure extremely stable and reproducible SERS signals. Single-molecule detection sensitivity utilizing the glycerol-stabilized 3D SERS hot spot matrix has been demonstrated by collecting the SERS spectra of dye molecules at a concentration of as low as 0.5 aM (5 x 10(-19) M) with a good signal-to-noise ratio. A long lifetime, ultrahigh SERS enhancement, and extremely stable peak shape make the 3D SERS hot spot matrix a sensitive, practical, and reliable tool for the detection and analysis of analytes at ultralow concentration or even at the single-molecule level in complex matrixes.