[Objective] The aim of this study was to characterize Vibro harveyi acyl-ACP synthetase (AasS) with free fatty acids of different chain length and other carboxylic acids as substrates in vitro. [Methods] We analyzed the catalytic activity of AasS in vitro qualitatively and quantitatively by Urea-PAGE and UV spectrophotometry, respectively. [Results] AasS could catalyze the synthesis of fatty acyl-ACPs using straight chain free fatty acids with different chain length as substrates, and the enzymatic activity was much higher than C6-C12 fatty acids. In the case of 3-hydroxyl fatty acids, AasS showed higher activities to C8-C14 chain length as substrates. Furthermore, AasS could utilize such as 20 amino acids, benzoic acid and salicylic acid as substrates, and all formed corresponding acyl-ACPs. [Conclusion] Vibro harveyi acyl-ACP synthetase (AasS) catalyzed different substrates with different activities, and this finding could provide a reference for analyzing the metabolism of amino acids.