3D highly ordered silver nanoparticles (AgNPs) coated silica photonic crystal beads (Ag/SPCBs) were prepared and exploited as a novel surface enhanced Raman scattering (SERS) substrate. The monodisperse and size-controlled SPCBs were prepared via self-assembly of silica nanoparticles process using a simple microfluidic device. Then the Ag/SPCBs were easily obtained by in situ growth of AgNPs onto the NH2-modified SPCBs. Field emitting scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDX) were used to characterize the Ag/SPCBs. The effect of silica nanoparticle size and AgNO3 concentration on the SERS performance of the resultant Ag/SPCBs substrate were discussed in detail. The results indicate that the Ag/SPCBs have highest SERS signals when silica nanoparticle size is 250 nm and AgNO3 concentration is 0.8 mg/mL. Using malachite green (MG) as model analyte, the Ag/SPCBs substrate displayed a high sensitivity and a wide linear range for MG. The well-designed Ag/SPCBs show high uniformity and excellent reproducibility, and can be used as an effective SERS substrate for sensitive assay application.