Herein, we report the formation of well-aligned ultrathin films of polyaniline-functionalized multiwalled carbon nanotubes (PANI@MWCNTs) with a high orientational order over a macroscopic area by Langmuir-Blodgett (LB) technique and its enhanced ammonia gas sensing properties. During the interfacial assembly process, the PANI@MWCNTs gradually align to form small ordered blocks at the air-water interface, which further organize as a well-defined oriented monolayer. The orientation and alignment of PANI@MWCNTs in Langmuir films at the air-water interface were systematically studied as a function of interface temperature using transmission electron microscopic analysis. Surface functionalization of MWCNTs with polyaniline was found to overcome the 3D aggregation of CNTs leading to an oriented assembly of PANI@MWCNTs. The formation and stability of the compact monolayer/multilayer structures of PANI@MWCNTs-based LB films have been extensively studied using a p-A isotherm analysis and thermodynamic approach. For the first time, such highly oriented LB films of PANI-functionalized MWCNTs have been employed for ammonia gas sensing applications at room temperature. The sensor was found to exhibit outstanding sensitivity toward NH3 at room temperature compared to random networks, which is attributed to the directed electron transport through the aligned PANI@MWCNTs. The ultrathin LB film allows fast analyte diffusion due to the adequate molecular accommodation in the oriented assembly of the active sensing layer. The large-scale alignment of PANI@MWCNTs demonstrated in this investigation would enable the fabrication of high-density MEMS (micro-electromechanical system)-based nanoscale sensor arrays for high-performance NH3 gas sensor applications.