Biosensors have received vigorous development and serious concern in recent years owing to the combinations with nanotechniques, flow-injection and micro-fluidics devices. Affinitive biosensor is a kind of high-tech sensing device based on the specific affinity between biological molecules, and the affinity of bioactive substance and substrate. With the advantages of high specificity, good sensitivity, high speed, low cost, on-line measuring and monitoring in complicated system, affinitive biosensors are widely used in biomedical fields, such as the detection of biomedical markers, nucleic acids, proteins, viruses, bacteria and toxins, the research of medicine actions mechanism, clinical drug screening, and etc. In this paper, the recent advance of affinitive biosensors in biomedical applications are reviewed. We also focus on a growing number of applications and progress in immunosensors and aptamer-based biosensors for tumor biomarker, nucleic acids and proteins. It covers the basic principles and biomedical and clinical applications of immunosensors and aptamerbased biosensors，and indicates the future prospects in this field.