The micellar characteristics of the three conventional surfactants such as sodium dodecyl sulfate (SDS), Hexadecyltrimethylammonium bromide (CTAB), and Triton X-100 (TX-100) are changed by the addition biologically active L-Glutamine (Glu) (i.e., 0.005, 0.01 and 0.02 M) were determined from stalagmometric, UV–visible spectroscopy, viscometric, FTIR and fluorescence methods using pyrene as probe. An observed decrease in I1/I3 in all micellar systems confirms the relative hydrophobicity of L-Glu. The CMCs values are reduced with the addition of L-Glu were determined by stalagmometric and it is confirmed by UV–visible and fluorescence methods and differs in the following order: SDS > CTAB > TX-100. The estimation of numerous interfacial parameters have also been done by using stalagmometric measurements including the (ϒCMC), (πCMC), (pC20), (Amin), and (Гmax). The concentration of L-Glu (M) rises, Amin values rise while the Гmax value falls and a significant rise in the surface tension of CMC (ϒCMC) values, with the values being in the range of 0.02 M > 0.01 M > 0.005 M > water. Hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waal interactions are only a few of the diverse types of interactions that were examined using FTIR in the pure surfactants and L-Glutamine mixed systems. To stabilize proteins and prevent protein aggregation, the findings presented here may be applied in pharmaceutical formulations.