FAME was produced by a two-step in-situ transesterification of acid oil (AO) with methanol in a novel continuous flow ultrasonic reactor system composed of four ultrasonic reactors with different frequency. The hydrodynamic behavior of the reactor was investigated by a step response technique, and the effect of ultrasonic frequency on mono-alkyl esters of long chain fatty acids (FAMEs) formation was also investigated. The production process includes an in-situ sulfuric acid-catalyzed esterification of AO with methanol in the first two ultrasonic reactors successively followed by an in-situ base-catalyzed transesterification in the other two ultrasonic reactors. The AO initial free fatty acids (FFA) content about 17.5 w% was cut down to less than 1 w% by sulfuric acid-catalyzed esterification. FAME yields in excess of 97.0% identified by gas chromatography/mass spectrometry (GC/MS) were obtained by the two-step in-situ reaction. The maximum and minimum volumetric productivity could reach 13.76 L·h-1 and 10.24 L·h-1 respectively.