In this study, we demonstrated a new approach for gas sensing using multilayer films self-assembled with cationic gold nanoparticles (AuNPs+) and anionic polyelectrolytes via layer-by-layer (LbL) deposition. The surface of AuNPs+ was modified with a cationic disulfide, [–S(CH2)2NHCO(CH2)10(CH3)3N+Br−]2. Two types of LbL film using AuNPs+ were self-assembled with anionic polyelectrolytes, poly(acrylic acid) (PAA), and poly(sodium 4-styrene sulfonate) (PSS) on quartz substrates, and their film growth was confirmed by UV–vis measurements. The 10-cycle AuNPs+/PAA film showed reproducible responses after exposure to ammonia gas (3000 ppm) and HCl treatment (0.1 M), showing absorbance changes of 0.0428 ± 0.0033 at 299 nm and 0.0053 ± 0.0013 at 544 nm for five repeated tests. On the other hand, no significant changes were observed for the 10-cycle AuNPs+/PSS film. Ammonia binding was based on the acid–base interaction between ammonia and the free carboxylic acid groups of PAA. The film's composition and morphology were also investigated by Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM) measurements to clarify the sensing mechanism.