Photoacoustic imaging (PAI), a novel imaging modality based on photoacoustic effect, shows great promise in biomedical applications. By converting pulsed laser excitation into ultrasonic emission, PAI combines the advantages of optical imaging and ultrasound imaging, which benefits rich contrast, high resolution and deep tissue penetration. In this paper, we introduced recent advances of contrast agents, applications, and signal enhancement strategies for PAI. The PA contrast agents were categorized by their components, mainly including inorganic and organic PA contrast agents. The applications of PAI were summarized as follows: deep tumor imaging, therapeutic responses monitoring, metabolic imaging, pH detection, enzyme detection, reactive oxygen species (ROS) detection, metal ions detection, and so on. The enhancement strategies of PA signals were highlighted. In the end, we elaborated on the challenges and provided perspectives of PAI for deep-tissue biomedical applications.