AbstractIR‐780 is a lipophilic dye with excellent optical and tumor imaging properties for early tumor diagnostics. Although the mechanism of tumor targeting has not been fully identified, the view that serum albumin plays an important role in tumor accumulation has been recognized. Here, the mechanism of the interaction between IR‐780 and HSA was studied to explore the effect of albumin on its tumor targeting properties. Data demonstrate that IR‐780 can be tightly adsorbed by HSA at a ratio of 1:1 to form a noncovalent complex, which exhibits significant improvement in the near‐infrared fluorescence imaging and tumor diagnosis capacity. During this process, the endogenous fluorescence and esterase activity of HSA are both partially inhibited by IR‐780, and the α‐helical content of HSA slightly increases. Molecular docking simulation displays that the binding site of IR‐780 on HSA is between subdomains IIA and IIB. These results indicate that HSA is an important factor to mediate the optical performance of IR‐780, giving it higher tumor diagnosis capability.