Determining glucose in biological samples is tedious and time-consuming due to sample pretreatment. The sample is usually pretreated to remove lipids, proteins, hemocytes and other sugars that interfere with glucose detection. A surface-enhanced Raman scattering (SERS) active substrate based on hydrogel microspheres has been developed to detect glucose in biological samples. Due to the specific catalytic action of glucose oxidase (GOX), the high selectivity of detection is guaranteed. The hydrogel substrate prepared by microfluidic droplets technology protects the silver nanoparticles from the surrounding environment and improves the stability and reproducibility of the assay. In addition, the hydrogel microspheres have size-adjustable pores that selectively allow small molecules to pass through. The pores block the entry of large molecules, such as impurities, enabling glucose detection through glucose oxidase etching without sample pretreatment. This hydrogel microsphere-SERS platform is highly sensitive and enables reproducible detection of different glucose concentrations in biological samples. The use of SERS to detect glucose provides clinicians with new diagnostic methods for diabetes and a new application opportunity for SERS-based molecular detection techniques.