According to recent research, amphiphilic surfactants can stabilize metal nanoparticles because of their excellent dispersibility. Therefore, herein, amphiphilic polyethylenimine (PEI) dendritic polymer nanocapsules were synthesized by using PEI and poly­(urea/malonamide) dendrons of different generations. Then, silver nanoparticles (AgNPs) were immobilized on PEI dendritic polymer nanocapsules for surface-enhanced Raman scattering (SERS) detection. Well-designed amphiphilic PEI dendrons possessed abundant nucleation sites for AgNP reduction, which could directly reduce silver ions to AgNPs without additional reducing agents at room temperature, showing the stronger reducing capability than pristine PEI. The size and interparticle gap of AgNPs were manipulated by varying the size of the PEI dendritic polymer and the ratio of PEI dendritic nanocapsules/AgNO3. As a result, the Ag-PEI-dendritic polymer was able to perform Raman enhancing capability. Especially, a sample, namely, Ag-PEI-G0.5, exhibited the strongest Raman enhancement effect, due to the optimal size and interparticle gap of AgNPs. The Ag-PEI dendritic SERS nanocapsules could rapidly and reproducibly detect many kinds of biomolecules (adenine, methylene blue, and beta-carotene) with a linear calibration curve. The limit of detection (LOD) of adenine was lower than 10–7 M. Therefore, this facile method to in situ prepare Ag-PEI dendritic SERS nanocapsules (reducing agent free) possesses high potential to be applied in rapid SERS detection for the quantitative analysis of biomolecules.