Photoacoustic imaging (PAI) has attracted great attention in the diagnosis and treatment of diseases due to its noninvasive properties. Especially in the second near-infrared (NIR-II) window, PAI can effectively avoid the interference of tissue spontaneous fluorescence and light scattering, and obtain high resolution images with deeper penetration depth. Because of its ideal spectral absorption and high conversion efficiency, NIR-II PA contrast agents overcome the absorption or emission of NIR-II light by endogenous biomolecules. In recent years, a series of NIR-II PA contrast agents have been developed to improve the performance of PAI in disease diagnosis and treatment. In this paper, the research progress of NIR-II PA contrast agents and their applications in biomedicine are reviewed. PA contrast agents are classified according to their composition, including inorganic contrast agents, organic contrast agents, and hybrid organic-inorganic contrast agents. The applications of NIR-II PA contrast agents in medical imaging are described, such as cancer imaging, inflammation detection, brain disease imaging, blood related disease imaging, and other biomedical application. Finally, the research prospects and breakthrough of NIR-II PA contrast agents are discussed.