The phenomenon of molecular recognition including further function is common in nature. Host-guest complexation occurring at Langmuir monolayers has been investigated in detail similarly to molecular recognition in solutions. Langmuir monolayers at the air-water interface provide a unique environment for molecular recognition. Characteristic binding efficiency and selectivity of water-soluble substrates by specific pairing of hydrogen bonding have been found for functionalized monolayers at the air-water interface. Hydrogen bonding network formation between a functionalized monolayer and a substrate enables cleavage of the head group of an amphiphile forming the monolayer by hydrolysis of a C=C double bond that links the head group to the hydrophobic tail. Molecular organization termed “molecular recognition-directed self-assembly” has been demonstrated through complementary base-pairing in monolayers at the air-water interface. Controlling of molecular arrangement of nucleobase amphiphiles in Langmuir monolayers has been attempted by using complementary base-pairing with sequence-designed oligonucleotides as a molecular template. This unique molecular recognition should be useful for nanotechnology.