Organic fluorescent materials (OFMs) play a crucial role in the development of biosensors, enabling the extraction of biochemical information within cells and organisms, extending to the human body. Concurrently, OFM biosensors contribute significantly to the progress of modern medical and biological research. However, the practical applications of OFM biosensors face challenges, including issues related to low resolution, dispersivity, and stability. To overcome these challenges, scientists have introduced interactive elements to enhance the order of OFMs. Highly-ordered assembled OFMs represent a novel material type applied to biosensors. In comparison to conventional fluorescent materials, highly-ordered assembled OFMs typically exhibit robust anti-diffusion properties, high imaging contrast, and excellent stability. This approach has emerged as a promising method for effectively tracking bio-signals, particularly in the non-invasive monitoring of chronic diseases. This review introduces several highly-ordered assembled OFMs used in biosensors and also discusses various interactions that are responsible for their assembly, such as hydrogen bonding, π–π interaction, dipole–dipole interaction, and ion electrostatic interaction. Furthermore, it delves into the various applications of these biosensors while addressing the drawbacks that currently limit their commercial application. This review aims to provide a theoretical foundation for designing high-performance, highly-ordered assembled OFM biosensors suitable for practical applications. Additionally, it sheds light on the evolving trends in OFM biosensors and their application fields, offering valuable insights into the future of this dynamic research area.