The S, N co-dopedcarbon dots was obtained through the one-step hydrothermal treatment of cystine and citric acid. The results suggested that the prepared carbon dot solution has fluorescence emission at 455 nm with an excitation wavelength of 350 nm. This fluorescent Carbon Dots (FCDs) showed stableemission performancein pH 6~11 and bright blueemission with a quantum yield of 61.7% and an average lifetime of 10.75 ns. In this article, a sample preparation scheme with self-assembled nanocomposite film of silver nanoparticle (AgN) was designed to enhanced the optically stimulated luminescence emission of FCDs. The enhance PL signal of FCDs grown on sensing film leading to a higher sensitivity of drug content detection. A uniform nanocomposite film substrate was prepared via the reduction in situ of silver ions in polydopamine (PDA). The Enhanced Fluorescence film FTO/PDA-AgN/PDDA/[PSS/PDDA]₃/FCDs was achieved by self-assembled the polyelectrolyte molecular Layer-by-Layer (LbL) on the nanocomposite film that was utilized to control the distance of silver nanoparticle (AgN) and fluorescent Carbon Dots (FCDs). The synchronous reduction method was easy to operate, and theresults of Ultraviolet, Fluorescence, Raman spectra and scanning electron microscopy revealed that the composite silver nanostructure in polydopamine film result in the AgN has the advantage of not being oxidized. The silver nanoparticles could enhance the optically stimulated luminescence emission of FCDs as the separation distance was optimally designed between FCDs and AgN surface, the fluorescent intensity of FCDs in SAMs increased by nearly 3 times with the corresponding fluorescence lifetime reduced from 6.08 to 2.98 ns. The characteristics of the AgN-enhanced fluorescence which has distance-dependence, accelerated radiation attenuation and correlation with the reduction degree of AgN were additional evidence for a local surface plasmon resonance effect between AgN and FCDs. The experimental results showed that the addition of puerarin (Pue) quenched the fluorescence signal of FCDs, and the degree of quenching had a good linear relationship with the content of puerarin in the range of 3.33×10^-7~1.50×10^-5 mol·L^-1. The linear regression equation is I₀/I=2.843×10⁴c_Pue+1.068, the correlation coefficient R=0.998 56, and the detection limit QL=2.31×10^-7 mol·L^-1. This established AgN-enhanced fluorescence system based on the SAMs could increase the sensitivity of the detection for puerarin as the detection limit was reduced about an order of magnitude.