Fluorescence biomarkers are crucial for understanding structure and dynamics of biological macromolecules. However, limitations in binding affinity and fluorescence response remain challenging for many existing markers. In this study, we synthesized a carbazole-rhodanine hybrid molecule (Cr[sbnd]Rh) and evaluated its potential as a fluorescent biomarker, focusing on its binding affinity and fluorescence behaviour upon interaction with serum proteins (bovine serum albumin (BSA) and human serum albumin (HSA)). The Cr[sbnd]Rh is amphiphilic and tends to interact with both hydrophobic and hydrophilic pockets of the serum proteins. The interactions between the Cr[sbnd]Rh and BSA, and as well as HSA, were investigated using various spectroscopic techniques and computational studies. The fluorescent biomarker Cr[sbnd]Rh exhibited incredibly high binding affinity for both BSA and HSA with a binding constant of ∼106 M−1. Fluorescence studies revealed that Cr[sbnd]Rh binding to BSA & HSA increases its fluorescence 28-fold and 15-fold respectively (QY: 0.0012 to 0.015). FRET studies revealed distinct energy transfer efficiencies of 67.9 % and 51.9 % for the Cr-Rh:BSA and Cr-Rh:HSA complexes, respectively, suggesting differing donor-acceptor distances that may arise from variations in the binding environments. Time-resolved fluorescence analysis revealed that the Cr-Rh:BSA complex displays a significantly enhanced lifetime of 0.7 ns, compared to the ∼200 ps lifetime of Cr[sbnd]Rh alone. CD results demonstrated alterations in the secondary structures of both BSA and HSA proteins in the presence of Cr[sbnd]Rh. Additionally, molecular docking studies and MD simulations further supported the mode of binding and stability of the complexes with the experimental studies. These findings highlight the potential of Cr[sbnd]Rh as a promising fluorescent biomarker for studying site-selective binding with BSA and HSA. Besides, the Cr[sbnd]Rh could be a valuable tool in protein interaction studies owing to its high binding affinity, significant fluorescence response, and specificity with proteins.