This paper describes the development of an automated, hand-held sensor for the fast assessment of seafood freshness. The sensor developed here combined: an array-based chemical sensor, composed of incrementally different conducting polymer elements deposited on a small chip; a highly sensitive, custom-made electronics for the detection of very small signal changes; precise temperature control of the sensor chamber; and an on-board microcontroller for data collection, storage, automation, and analysis. The instrument was used to successfully test seafood samples with different degree of freshness and spoilage. A linear relationship between microbiological count and e-Nose signal for three different fish fillet was developed. Once the linear relationship is included into the hand-held unit software, the e-Nose signal can be used for assessment of seafood freshness without performing the microbiological count technique.