A novel colorimetric method based on polyaniline (PANI) film for the development of smart packaging, as a chemical sensor for real-time monitoring of the microbial breakdown products in the headspace of packaged fish is described. This on-package indicator contains PANI film, that responds through visible color change to a variety of basic volatile amines (specifically known as total volatile basic nitrogen (TVBN)) released during fish spoilage period. The PANI film characteristics and its response to TVBN were studied. A kinetic approach was used to correlate the ammonia response of the PANI film to that of the fish spoilage. Color changes, in terms of total color difference of PANI, correlated well with TVBN levels of fish. Apart from TVBN, trials on milkfish sample (Chanos chanos) have verified that the PANI film response also correlates well with microbial growth patterns in fish samples, especially the changing microbial populations (total viable count (TVC) and Pseudomonas spp.). These responses enabled the real-time monitoring of fish spoilage either at various constant temperatures or with temperature fluctuations. The PANI film can be recycled several times using an acid solution to regenerate the PANI surface. Thus, PANI film can be considered as a low-cost sensor suitable for smart packaging applications.