A first-of-a-kind latent electrochemical redox probe, ferrocene carbamate phenyl acrylate (FCPA), was developed for the selective detection of cysteine (Cys) and aminoacylase (ACY-1). The electrochemical signal generated by this probe was shown to be highly specific to Cys and insensitive to other amino acids and biological redox reactants. The FCPA-incorporated electrochemical sensor exhibited a broad dynamic range of 0.25-100 μM toward Cys. This probe also proficiently monitored the ACY-1-catalyzed biochemical transformation of N-acetylcysteine (NAC) into Cys, and this proficiency was used to develop an electrochemical assay for quantifying active ACY-1, which it did so in a dynamic range of 10-200 pM (0.1-2 mU/cm3) with a detection limit of 1 pM (0.01 mU/cm3). Furthermore, the probe was utilized in real-time tracking and quantification of cellular Cys production, specifically in Escherichia coli W3110, along with a whole blood assay to determine levels of Cys and spiked ACY-1 in blood with a reliable analytical performance.