Developments during the past decade in gradient biomaterials have resulted in a tremendous upsurge of interest in generating spatially and temporally complex cell microenvironmental features at different length scales. Micro- and nanotechnological innovations have enabled the manipulation of such gradient biomaterials suitable for numerous biomaterial applications including basic cell studies, drug screening, and tissue engineering. Gradient biomaterials have recently been used to repair or regenerate complex tissues such as soft to hard interface tissues, which fail to recover their function through conventional tissue engineering approaches used for homogenous tissues. In this regard, there is a great need for the development of improved biomaterial systems with gradient in material composition and properties that encourage the differentiation of multiple cell phenotypes and tissue development, which can help engineering more complex tissues. The purpose of this review is to describe various gradient biomaterials employed in tissue engineering, their methods of fabrication, and challenges that must be met in order for them to reach their full potential. This review is expected to be useful for readers to gain knowledge on the state-of-the-art of tissue engineering using gradient biomaterials. © 2011 American Scientific Publishers. All rights reserved.