Macrophages are an important component of host defense and inflammation and play a pivotal role in immune regulation, tissue remodeling, and metabolic regulation. Since macrophages are ubiquitous in human bodies and have versatile physiological functions, they are involved in virtually every disease, including cancer, diabetes, multiple sclerosis, and atherosclerosis. Molecular biological and histological methods have provided critical information on macrophage biology. However, many in vivo dynamic behaviors of macrophages are poorly understood and yet to be discovered. A better understanding of macrophage functions and dynamics in pathogenesis will open new opportunities for better diagnosis, prognostic assessment, and therapeutic intervention. In this article, we will review the advances in macrophage tracking and analysis with in vivo optical imaging in the context of different diseases. Moreover, this review will cover the challenges and solutions for optical imaging techniques during macrophage intravital imaging.