Immobilization of biomaterials developed rapidly due to the great promise in improving their stability, activity and even selectivity. In this review, the immobilization strategies of biomaterials, including physical adsorption, encapsulation, covalent attachment, cross-linking and affinity linkage, were briefly introduced. Then, the major emphasis was focused on the reported various types of immobilized biomaterials, including proteins, enzymes, cell membrane and artificial membrane, living cells, carbohydrates and bacteria, used in the herbal analysis for bioactive compound screening, drug-target interaction evaluation and chiral separation. In addition, a series of carrier materials applied in biomaterials immobilization, such as magnetic nanoparticles, metal-organic frameworks, silica capillary column, cellulose filter paper, cell membrane chromatography, immobilized artificial membrane chromatography and hollow fiber, were also discussed. Perspectives on further applications of immobilized biomaterials in herbal analysis were finally presented.