Viruses are changing the biosensing and biomedicine landscape due to their multivalency, orthogonal reactivities, and responsiveness to genetic modifications. As the most extensively studied phage model for constructing a phage display library, M13 phage has received much research attention as building blocks or viral scaffolds for various applications including isolation/separation, sensing/probing, and in vivo imaging. Through genetic engineering and chemical modification, M13 phages can be functionalized into a multifunctional analysis platform with various functional regions conducting their functionality without mutual disturbance. Its unique filamentous morphology and flexibility also promoted the analytical performance in terms of target affinity and signal amplification. In this review, we mainly focused on the application of M13 phage in the analytical field and the benefit it brings. We also introduced several genetic engineering and chemical modification approaches for endowing M13 with various functionalities, and summarized some representative applications using M13 phages to construct isolation sorbents, biosensors, cell imaging probes, and immunoassays. Finally, current issues and challenges remaining in this field were discussed and future perspectives were also proposed.