An amino acid analyzer method for the simultaneous determination of 20 free amino acids (FAAs) and glutathione (GSH) in Penaeus vannamei (PV), Penaeus vannamei, Penaeus hidulis (PH) and Penaeus japonicus (PJ) were developed. The effects of different concentrations of trichloroacetic acid (TCA) and ethanol on the extraction of free amino acids were investigated, and 120 g·L-1 TCA was found to be ideal. The target analytes were eluted in sodium citrate buffer B1 (pH = 3.3) containing 135 mL·L-1 ethanol and 1 mol·L-1 sodium hydroxide (7 mL) and at the optimizing conversion time of sodium citrate buffer B2 (pH = 3.2) and sodium citrate buffer B3 (pH = 4.0) of 5.6 min, and the effective separation was achieved within 29.5 min. The developed method showed good linearity (R2 ≥ 0.9991) in the range of 1-250 µg·mL-1 with good intra-day and inter-day precision (relative standard deviations ≤ 2.38%) and spike recovery (86.42-103.64%). GSH and cysteine were used to identify marine prawn and freshwater shrimp. Hydroxyproline and serine were used to distinguish PV and Macrobrachium nipponense (MN) from others, respectively. The highest content of the total FAAs was found in PV, and principal component analysis revealed that PV had the highest comprehensive score for FAAs and GSH. Arginine was found to have the greatest influence on shrimp flavor.