Understanding the fundamentals of confined chemical reaction was an important subject in various heterogeneous physicochemical processes. Here, we investigated the orientation behavior of an amphiphilic ligand, the tri-n-octylphosphine oxide (TOPO), in a compressed monolayer at the air/water interface and its impact on the complexation reactivity of TOPO molecules with chromate ions at the interface. The analysis of sum frequency generation and polarization modulation infrared reflection absorption spectroscopy experiments combined with surface pressure measurements reveals a significant dependence of the adsorption rate and saturated concentration of chromate ions on the orientation of TOPO molecules during the increase of the surface pressure. In parallel, the analysis of molecular dynamics simulations indicates that the interaction energy between TOPO molecules and chromate ions is strongly sensitive to the orientation of TOPO molecules confined at the water/air interface. The present work provides a novel insight into the unique chemical reactivity of molecules in a confined microenvironment, and it provides a basis for further progresses in improving chemical reactivity and selectivity of molecules in a confined environment by regulating confinement of molecules.