Being perspective candidates for bioapplications, gemini surfactant – protein interaction research is continuing extensively. In the work described herein, we have, therefore, investigated the binding of a recently synthesized biodegradable cationic diester-functionalized C14-E2O2-C14 gemini surfactant with ethylene oxide group in the spacer with the transport protein bovine serum albumin (BSA). The system properties were determined through a combination of surface tension measurements, multispectroscopic and computational analyses. The surface and interfacial properties of the concerned gemini got perturbed on interaction with BSA. The reduction in BSA fluorescence intensity was accredited to fluorescence quenching by the surfactant and relevant parameters (KSV and Kb; 14.70, 10.63, 7.27 × 104 M−1 and 25.63, 14.28, 7.20 × 104 M−1) have been computed at three different temperatures (288, 298 and 308 K), which demonstrated that C14-E2O2-C14 binds strongly with BSA protein through static quenching (UV–vis absorption analysis results were the same) with the binding stoichiometry 1:1. The obtained negative values of thermodynamic parameters (ΔGb0,ΔHb0 and ΔSb0) specified spontaneous process where hydrogen bonding and van der Waals forces are the major forces in the interaction mechanism. The effect of β-cyclodextrin (β-CD) has also been explored on the interaction and the values of KSV and Kb have been evaluated. Moreover, some other spectral results (CD, 3-D, synchronous, RRS, pyrene, FT-IR) confirmed that the interaction of the surfactant with BSA persuaded alterations in conformation of BSA. The molecular docking results as well as site probe measurements corroborated the ligand binding sites and interaction of the gemini surfactant with amino acid residues. The binding analysis of C14-E2O2-C14 gemini surfactant-BSA interaction is potentially valuable for elucidating chemico-biological interaction that can be used in the pharmaceuticals, biochemistry and pharmacology arenas.