Langmuir monolayers formed by dipalmityl phosphatidylcholine (DPPC) molecules at the air/liquid interface are an important model system for studying the biomembranes, and their chiral structure and interaction with foreign substances have been the frontiers of related fields. Vitamin B₂ (VB₂) is an important medicine for treating lipid deposition diseases caused by metabolic disorders. The molecular mechanism of the interaction between VB₂ and phospholipids is the key to understanding the function of VB₂. In this report, the interaction between VB₂ and DPPC at the air/water interface was studied using second-harmonic generation linear dichroism (SHG-LD), Langmuir membrane balance and Brewster Angle Microscopy (BAM). The compression isotherm shows that the surface pressure in the liquid expansion phase/liquid condensed phase (LE/LC) of L-DPPC and D-DPPC at the water interface is almost unchanged, while the film pressure region in the co-existing phase of race-DPPC is slightly shortened. The LE /LC coexistence phase of race-DPPC disappears at the interface of the VB₂ aqueous solution. In addition, the elastic modulus studies show that the VB₂ molecule can increase the elastic modulus of L-DPPC monolayers, but reduce the elastic modulus of D-DPPC and race-DPPC monolayers. SHG-LD results showed that the Degree of Chiral Excess (DCE) of L-DPPC at the interface between VB₂ and water was unchanged at the surface pressure of 13 mN·m^-1, and the DCE of D-DPPC at the interface between VB₂ and water was reversed. The DCE value of race-DPPC does not change with the change of subphase. Under the same surface pressure, BAM observed that the interaction of L-DPPC and D-DPPC to assemble into homochiral microdomains with different arm curvature directions at the pure water interface, and VB₂ could induced the increase of D-DPPC microdomains by 1 to 2 times. Meanwhile, VB₂ induced the extension of approximately circular microdomains on race-DPPC monolayer and the growth of three curvature arms, which could be explained as VB₂ reducing the energy of non-monolayer chiral interactions and resulting in chiral phase separation. VB₂ also induced changes in the chiral structure of race-DPPC monolayers. This study is of great significance for understanding the regulation of VB₂ on the macrostructure and biological function of the phospholipid membrane.