Macrophage is a kind of immune cell and performs multiple functions including pathogen phagocytosis, antigen presentation and tissue remodeling. To fulfill their functionally distinct roles, macrophages undergo polarization towards a spectrum of phenotypes, particularly the classically activated (M1) and alternatively activated (M2) subtypes. However, the binary M1/M2 phenotype fails to capture the complexity of macrophages subpopulations in vivo. Hence, it is crucial to employ spatiotemporal imaging techniques to visualize macrophage phenotypes and polarization, enabling the monitoring of disease progression and assessment of therapeutic responses to drug candidates. This review begins by discussing the origin, function and diversity of macrophage under physiological and pathological conditions. Subsequently, we summarize the identified macrophage phenotypes and their specific biomarkers. In addition, we present the imaging probes locating the lesions by visualizing macrophages with specific phenotype in vivo. Finally, we discuss the challenges and prospects associated with monitoring immune microenvironment and disease progression through imaging of macrophage phenotypes.