Nisin is a bacteriocin produced by Streptococcus and Lactococcus species and has antimicrobial activity against other bacteria. Nisin omits the need to use chemical preservatives in food due to its biological preserving properties. In the present in vitro study, we investigated nisin interaction with bovine serum albumin (BSA) using fluorescence spectroscopy and surface plasmon resonance (SPR) analysis to obtain information about the mechanisms of BSA complex formation with nisin. The BSA fluorescence intensity values gradually diminished with rising nisin concentration. The BSA fluorescence quenching analysis indicated that a combined quenching mechanism plays the main role. Finally, the Kb values were reduced with increasing temperature, which is demonstrative of nisin-BSA complex stability decrease at high temperatures. The negative values of ΔH° and ΔS° showed that hydrogen bonds and van der Waals forces are the foremost binding force between BSA and nisin. Meanwhile, the negative values of ΔG° demonstrated the exothermic and random nature of the reaction process. The results of the SPR verified the gained results through the fluorescence spectroscopy investigation, which denoted that the BSA affinity to nisin diminished upon increasing temperature. Overall, fluorescence spectroscopy and SPR results showed that the BSA interaction with nisin decreased with rising temperatures.