Polymeric microspheres are used in medical diagnostics, therapy, and as supports for enzymes and other proteins in biotechnology. These particles have also found application in fundamental studies in the life-sciences as useful tools for separation of the living cells and subcellular fragments. This article presents comprehensive information on polyacrolein containing microspheres suitable for medical applications. Various synthetic procedures (radical, anionic, and red-ox polymerizations) are described, leading to polyacrolein microspheres, microspheres made of polyacrolein and other polymers, and copolymer microspheres containing polyacrolein segments. These particles can be obtained in a range of diameters from ca. 50 nm to a few micrometers, often with very narrow diameter distributions. Depending on the particular synthetic routes, it is possible to obtain microspheres with a uniform structures, core-shell, and raspberry morphologies. In this review, methods used for the characterization of the chemical structure of the surface layer of microspheres, in particular, methods used for determination of reactive (aldehyde) groups are also discussed. Results of studies on the immobilization of proteins onto polyacrolein containing microspheres, including relationships between the mode of protein binding (covalent immobilization and physical adsorption) and the fraction of polyacrolein in the surface layer of microspheres are reviewed. Methods allowing one to obtain protein-microsphere systems in which the attached proteins retain a substantial part of their biological activity are described. Polyacrolein containing microspheres, after attachment of selected antibodies, can be used as reagents in diagnostic tests. There are examples of using these particles for the fabrication of new materials for hemoperfusion columns and for the formation of two-dimensional particle assemblies on surfaces of polyethylene, quartz, and glass, which are potentially useful for the fabrication of biosensors.