The authors describe an amperometric biosensor for the amino acid L-arginine (L-Arg). It is based on the use of a Nafion/Polyaniline (PANi) composite on a platinum screen-printed electrode (Pt-SPE) using a novel recombinant arginine deiminase isolated from Mycoplasma hominis. The protein was over-expressed, purified and employed as a biorecognition element of the sensor. Enzymatic hydrolysis of L-Arg leads to the formation of ammonium ions which diffuse into the Nafion/PANi layer and induce the electroreduction of PANi at a potential of -0.35 V (vs Ag/AgCl). L -Arg sensitivity was 684±32 A⋅M-1⋅m-2, and the apparent Michaelis-Menten constant (KMapp) 0.31±0.05 mM. The calibration plot was linear over the range 3 - 200 μM L-Arg, the limit of detection was 1 μM, and the response time (for 90% of the total signal change to occur) 15 s. The sensor was selective and exhibited good storage stability (> 1 month without loss in signal). The biosensor was applied to the analysis of L-Arg in pharmaceutical samples and of ammonium and L-Arg in spiked human plasma obtained from blood of healthy volunteers and those with a hepatic disorder. Data generated was found to be in good agreement with a reference fluorometric enzymatic assay.