The worldwide abuse of illicit drugs led to severe consequences for human health, and society environment. Therefore, urgently required are effective and efficient on-site detection methods for illicit drugs of interest in various matrices, e.g., police samples, biofluids, and hairs. Although surface-enhanced Raman spectroscopy (SERS) shows power in many analytical fields, the cumbersome pretreatment of various matrices restricts its use in the easy-to-operate and on-site detection of illicit drugs. To address this problem, we adopted pore-size selectivity SERS-active hydrogel microbeads, whose meshes are adjustable to allow small molecules to access and to exclude large molecules. Meanwhile, Ag nanoparticles were uniformly dispersed and wrapped in the hydrogel matrix, providing excellent SERS performances with high sensitivity, reproducibility, and stability. By using these SERS hydrogel microbeads, one of the illicit drugs, methamphetamine (MAMP), can be rapidly and reliably detected in various biological specimens (blood, saliva, and hair) without sample pretreatment. The minimum detectable concentration is 0.1 ppm for MAMP in three biological specimens with a linear range of 0.1-100 ppm, which is lower than the maximum allowable level of 0.5 ppm set by the department of the health and human service. The SERS detection results were consistent with the gas chromatographic (GC) data. Thanks to its operational simplicity, fast response, high throughput and low cost, our established SERS hydrogel microbeads can be used as a sensing platform for facile analysis of illicit drugs through simultaneous separation, preconcentration, and optical detection, which shall be provided practically for front-line narcotics squad and resistance to the overwhelmed drug abuses.