Alopecia is globally recognized as a formidable therapeutic challenge, impacting both physiological health and psychological well-being. The crux of effective treatment lies in achieving de novo hair follicle neogenesis, a process that transcends merely stimulating existing follicles. Research into hair follicle organoids (HFOs) is advancing rapidly, transitioning from rudimentary self-assembly models toward high-fidelity, clinical application-driven paradigms. To provide a rigorous synthesis of the current landscape, this review conducted a systematic literature search across the Web of Science, PubMed, and Google Scholar databases. The search strategy utilized combinations of key terms, including “hair follicle organoid,” “alopecia,” “hydrogel,” and “3D bioprinting,” spanning the last 15 years. We comprehensively summarized the design principles and recent breakthroughs in HFO technology. First, the biological foundations of hair follicle development and the specific requirements of its inductive microenvironment were elucidated. Subsequently, we highlighted design strategies for functionalized hydrogels to simulate the hair follicle niche. This included a detailed discussion on modulating physicochemical properties and integrating advanced manufacturing technologies, such as three-dimensional bioprinting. Finally, the potential of HFOs in high-throughput drug screening and complex wound repair was assessed. By serving as a robust, human-relevant in vitro model, HFOs can significantly reduce reliance on animal testing and accelerate the discovery of hair-growth-promoting compounds. By providing both theoretical frameworks and technical insights, this review aims to support the development of high-performance hair follicle regeneration platforms and accelerate their transition from laboratory research to clinical translation.