Noraucuparin, a biphenyl phytoalexin, emerges as a promising drug candidate. Serum albumin plays a very important role in the in vivo transportation of drugs. The investigation delves into the binding interaction between noraucuparin and BSA employing comprehensive spectroscopic methods and molecular docking technique. UV–vis and FL spectrum results indicated that noraucuparin bond with BSA, and there was a new complex produced at the binding site II in domains IIIA (Ser-453 residue). The dissociation constants Kd from The ITC analysis was found to be 4.63 × 10−5 M at 298 K and 6.67 × 10−5 M at 310 K, showing 310 K is favorable for the combination of noraucuparin and BSA. The negative enthalpy (ΔH) and entropy (ΔS) values for the interaction revealed that the binding behavior was driven by hydrogen bonds. The results obtained from 3D fluorescence and CD spectrum illustrated that noraucuparin could change the secondary structure of BSA. According to molecule docking result, the binding energy of interaction is −7.09 kcal·mol−1. These revelations furnish a nuanced molecular-level comprehension of noraucuparin's mechanism of action on serum albumin. The discernments garnered from this study bear substantial reference significance and practical implications for ongoing lead compound exploration in antibiotics, particularly in vivo contexts.