Hydrogel microspheres (HMs) are versatile biomaterials with biocompatibility and controlled release properties, widely applied in drug delivery, cell carriers, and tissue engineering. Their tunable material compositions (natural, synthetic, or composite polymers) and diverse fabrication techniques (e.g., microfluidics, electrohydrodynamic spraying) allow precise regulation of size, morphology, and functionality, supporting applications from musculoskeletal repair to dermatological therapy. Despite rapid advancements, a comprehensive understanding of HM design, manufacturing, and biomedical applications is still lacking, as existing reviews mainly focus on single fields or specific scenarios. This review systematically summarizes HMs construction strategies (material selection and property modulation), fabrication technologies (batch emulsion, microfluidic chips, and emerging Artificial Intelligence (AI)-assisted methods), and multifunctional applications (drug and cell delivery, nanoparticle integration, and lubrication modification). It highlights the cross-system therapeutic potential of HMs and discusses challenges in clinical translation. By integrating these aspects, this review aims to bridge the gap between material design and clinical translation, providing researchers with an overview from basic research to clinical application, while exploring approaches to cross-system synergistic therapy and addressing bottlenecks in clinical translation.