Nitrogen-containing heterocyclic compounds represent fundamental structural elements in contemporary medicinal chemistry. Pyrrolodiazepines, which result from the fusion of pyrrole and diazepine rings, integrate the beneficial properties of both parent structures and have emerged as a significant area of research in synthetic chemistry and pharmaceutical development in recent years. This review provides a systematic overview of the synthetic methodologies for pyrrolodiazepine derivatives that have been developed recently, encompassing transition-metal catalysis, cascade cyclization, multicomponent reactions, photoredox catalysis, base-mediated cyclization, and environmentally friendly synthesis approaches. The advantages and limitations of each method are critically evaluated. Furthermore, the biological activities of pyrrolodiazepines are extensively reviewed, addressing their antitumor, antibacterial, antiviral, central nervous system regulatory, metabolic, and cardiovascular effects, along with corresponding structure–activity relationship analyses. Finally, the review identifies the current challenges in the synthesis and application of pyrrolodiazepines and explores potential future development directions.