It is important to elucidate molecular structures of polymer and biological molecules at interfaces because such structures mediate functions of these molecules. However, it is difficult to examine such interfaces in situ in real time due to the lack of appropriate tools. In the recently years, we have used a nonlinear optical laser spectroscopy, sum frequency generation (SFG) vibrational spectroscopy, to investigate molecular structures of interfacial polymer and biological molecules in situ. We have successfully elucidated interfacial molecular structures of polymer materials, antimicrobial peptides, membrane associated proteins, and various enzymes using SFG, supplemented by attenuated total reflectance (ATR)-FTIR and circular dichroism (CD) spectroscopy as well as all-atom and coarse-grain molecular dynamics simulations. This talk will focus on our recent results on semiconducting polymers, surface immobilized peptides and enzymes, and molecular interactions between 2D materials and biological molecules.