In this article, we explored the surface chemistry properties of a cholera toxin B (CTB) monolayer at the air subphase interface and investigated the change in interfacial properties through in situ spectroscopy. The study showed that the impact of the blue shift was negligible, suggesting that the CTB molecules were minimally affected by the subphase molecules. The stability of the CTB monolayer was studied by maintaining the constant surface pressure for a long time and also by using the compression decompression cycle experiments. The high stability of the Langmuir monolayer of CTB clearly showed that the driving force of CTB going to the amphiphilic membrane was its amphiphilic nature. In addition, no major change was detected in the various in situ spectroscopy results (such as UV vis, fluorescence, and IR ER) of the CTB Langmuir monolayer with the increase in surface pressure. This indicates that no aggregation occurs in the Langmuir monolayer of CTB.