We have synthesized a new gemini surfactant, glycol bis-N-tetradecyl nicotinate dibromide (designed as P14-1-14), and probed its combination with bovine serum albumin (BSA) by fluorescence spectroscopy, dual polarization interferometry (DPI), proton nuclear magnetic resonance spectroscopy ( 1 H NMR), Fourier transform infrared spectroscopy (FTIR) and molecular docking. The experiments revealed, in one hand, P14-1-14 quenched the intrinsic fluorescence of BSA by way of forming BSA/P14-1-14 complex with a binding constant of 2.14×10 4 Lmol −1 at 293K, that P14-1-14 augmented the thickness, mass, the refractive index (RI) and density of BSA, and that P14-1-14 declined the contents of α-helix (from 54.01% to 29.41%) but raised the random structure (from 7.86% to 22.27%). On the other hand, BSA shifted the proton resonance signals of P14-1-14 up-field and reduced relaxation time. The molecular docking manifested P14-1-14 embedded into subdomain of BSA, which made possible the π–π stacking between the electron-deficit pyridinium rings in P14-1-14 and the electron-abundant pyrrole ring in Trp residues of BSA, and that hydrogen bonding and hydrophobic interaction also took place during the binding. Therefore, the present work offers a whole view of the interaction of BSA with a new gemini surfactant.